Antibody that binds to VEGF and IL-1beta and methods of use

ABSTRACT

The present invention relates to anti-VEGF/anti-IL-1beta antibodies and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 16/722,317, filed Dec. 20, 2019, which claims benefit of priority toEuropean Patent Application No. 18215023.5 filed Dec. 21, 2018, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jul. 20, 2021, is namedP35223-US-1_SeqList.txt and is 26,383 bytes in size.

FIELD OF THE INVENTION

The present invention relates to anti-VEGF/anti-IL-1beta antibodies andmethods of using the same.

BACKGROUND OF THE INVENTION

A bispecific antibody binding to IL-1beta and VEGF has been reportedpreviously and was suggested for treatment of ocular vascular diseases(WO2016/075034, antibody “0032”). The bispecific anti-VEGF/anti-IL-1betaantibody 0032 is a full length IgG-like antibody with a VH/VL domainexchange in one binding arm (WO2009/080252, Schaefer, W. et al, PNAS,108 (2011) 11187-1191), wherein the binding arm of the wild typeantibody domain arrangement specifically binds to IL-1beta and thebinding arm comprising the VH/VL domain crossover specifically binds toVEGF. The VEGF binding arm comprises the VH and VL domains of anti-VEGFantibody ranibizumab.

Multispecific antibodies comprising two paratopes in one pair of avariable heavy chain domain (VH) and a variable light chain domain (VL)have been described in WO2008/027236; WO2010/108127 and Bostrom, J., etal., Science 323 (2009) 1610-1614 as well as in WO2012/163520.

WO2012/163520 discloses bispecific antibodies comprising twonon-overlapping paratopes in one pair of VH and VL domains (“DutaFabs”).Each paratope of the bispecific antibody of WO2012/163520 comprisesamino acids from the heavy chain and from the light chain CDRs, whereinheavy chain CDR-H1 and CDR-H3 as well as light chain CDR-L2 contributeto the first paratope and light chain CDR-L1 and CDR-L3 as well as heavychain CDR-H2 contribute to the second paratope Monospecific antibodiescomprising the individual paratopes are isolated independently fromdifferent Fab-libraries, which are diversified in either the first orthe second paratope. The amino acid sequences of said monospecificantibodies are identified and merged into the biparatopic VH and VLpair. One exemplary Fab fragment specifically binding to VEGF and IL-6is disclosed in WO2012/163520.

There is a need for improved therapeutic antibodies that bind to VEGFand IL-1beta.

SUMMARY OF THE INVENTION

The present invention relates to bispecific anti-VEGF/anti-IL-1betaantibodies and methods of using the same.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a VEGF paratope and an IL-1betaparatope within one cognate pair of a variable light chain domain (VLdomain) and a variable heavy chain domain (VH domain), wherein the VEGFparatope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 ofthe antibody, wherein the IL-1beta paratope comprises amino acidresidues from the CDR-H1, CDR-H3 and CDR-L2 of the antibody.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a VEGF paratope and an IL-1betaparatope within one cognate pair of a VL domain and a VH domain, whereinthe pair of the variable light chain domain and the variable heavy chaindomain simultaneously binds to human VEGF and human IL-1beta.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a VEGF paratope and an IL-1betaparatope within one cognate pair of a VL domain and a VH domain, whereinnone of the amino acids that are comprised in the VEGF paratope arecomprised in the IL-1beta paratope.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a VEGF paratope and an IL-1betaparatope within one cognate pair of a VL domain and a VH domain, whereinthe antibody binds to the same epitope on human VEGF and to the sameepitope on human IL-1beta as an antibody with a variable heavy chaindomain of SEQ ID NO: 11 and a variable light chain domain of SEQ ID NO:12.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein an antibody Fab fragment of theantibody binds (i) to human VEGF121 with a K_(D) of less than 10 pM asmeasured by surface plasmon resonance, and (ii) to human IL-1beta with aK_(D) of less than 30 pM as measured by surface plasmon resonance.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein an antibody Fab fragment of theantibody exhibits an aggregation onset temperature of more than 70° C.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein an antibody Fab fragment of theantibody exhibits a melting temperature of more than 80° C. as measuredby dynamic light scattering.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein binding of an antibody Fab fragmentof the antibody to human VEGF inhibits binding of VEGF to VEGFR2 with anIC50 of less than 50 nM as measured by surface plasmon resonance; andwherein binding of an antibody Fab fragment of the antibody to humanIL-1beta inhibits binding of IL-1beta to IL-1betaR1 with an IC50 of lessthan 30 nM as measured by surface plasmon resonance.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, (d) ahuman heavy chain framework with (i) FR1 comprising amino acid residuesE2, G26, V28, and K30, (ii) FR3 comprising amino acid residues R66, R83,and K94; and a VL domain comprising (e) CDR-L1 comprising the amino acidsequence of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence of SEQ IDNO:8, and (h) a human light chain framework with (i) FR1 comprisingamino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii)FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a VH domain comprising amino acidresidues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61,K62, F63, I64, R66, R83, K94, D95, V96, F98, and D101, and a VL domaincomprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50,Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94, and Y96, whereinthe numbering of the VH and VL domains is according to the Kabatnumbering system. In one embodiment the antibody comprises a VEGFparatope comprising the following amino acid residues in the VH domainD55, H56, Y58, T61, K62, F63, I64, R66, and R83, and the following aminoacid residues in the VL domain 12, Y27, W27a, S27c, S27d, E67, D68, Q69,R92, Y93, H94, and Y96; and an IL-1beta paratope comprising thefollowing amino acid residues in the VH domain E2, G26, V28, K30, W31,N35b, D35c, K52a, K94, D95, V96, F98, and D101, and the following aminoacid residues in the VL domain L32, Y49, D50, Y53, K54, L56, G57, Y91.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a VH domain comprising amino acidresidues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61,K62, F63, 164, R66, R83, K94, D95, V96, F98, and D101, and a VL domaincomprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50,Y53, K54, L56, G57, S67, H68, E69, Y91, R92, Y93, H94, and Y96, whereinthe numbering of the VH and VL domains is according to the Kabatnumbering system. In one embodiment the antibody comprises a VEGFparatope comprising the following amino acid residues in the VH domainD55, H56, Y58, T61, K62, F63, I64, R66, and R83, and the following aminoacid residues in the VL domain I2, Y27, W27a, S27c, S27d, S67, H68, E69,R92, Y93, H94, and Y96; and an IL-1beta paratope comprising thefollowing amino acid residues in the VH domain E2, G26, V28, K30, W31,N35b, D35c, K52a, K94, D95, V96, F98, and D101, and the following aminoacid residues in the VL domain L32, Y49, D50, Y53, K54, L56, G57, Y91.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising (a) a VH domain comprising anamino acid sequence having at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:11; and (b) a VL domain comprising an aminoacid sequence having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:12.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:11;and (b) a VL domain comprising an amino acid sequence having at least90% sequence identity to the amino acid sequence of SEQ ID NO:12.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:11,wherein the VH domain comprises amino acid residues E2, G26, V28, K30,R66, R83, and K94; and (b) a VL domain comprising an amino acid sequencehaving at least 90% sequence identity to the amino acid sequence of SEQID NO:12, wherein the VL domain comprises amino acid residues I2, Y49,G57, E67, D68, and Q69, wherein the numbering of the VH and VL domainsis according to the Kabat numbering system.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, (d) ahuman heavy chain framework with (i) FR1 comprising amino acid residuesE2, G26, V28, and K30, (ii) FR3 comprising amino acid residues R66, R83,and K94; and a VL domain comprising (e) CDR-L1 comprising the amino acidsequence of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence of SEQ IDNO:8, and (h) a human light chain framework with (i) FR1 comprisingamino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii)FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem, comprising (a) a VH domain comprising an amino acid sequencehaving at least 90% sequence identity to the amino acid sequence of SEQID NO:11; and (b) a VL domain comprising an amino acid sequence havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:12.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising (a) a VH domain comprising anamino acid sequence of SEQ ID NO:11 with up to 15 amino acidsubstitutions; and (b) a variable light chain domain comprising an aminoacid sequence of SEQ ID NO:12 with up to 15 amino acid substitutions. Inone embodiment the antibody comprises (a) a VH domain comprising anamino acid sequence of SEQ ID NO:11 with up to 15 amino acidsubstitutions, wherein the amino acid substitutions are located atpositions 3 to 25, 36 to 49, 97 to 82c, 84 to 93, or 103 to 113 of SEQID NO:11; and (b) a variable light chain domain comprising an amino acidsequence of SEQ ID NO:12 with up to 15 amino acid substitutions, whereinthe amino acid substitutions are located at positions 1, 4, 6, 8 to 23,35 to 48, 58 to 66, 70 to 88, or 98 to 107 of SEQ ID NO:12, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO:11 with up to 15 amino acid substitutions; and (b) a variable lightchain domain comprising an amino acid sequence of SEQ ID NO:12 with upto 15 amino acid substitutions.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, (d) ahuman heavy chain framework with (i) FR1 comprising amino acid residuesE2, G26, V28, and K30, (ii) FR3 comprising amino acid residues R66, R83,and K94; and a VL domain comprising (e) CDR-L1 comprising the amino acidsequence of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence of SEQ IDNO:8, and (h) a human light chain framework with (i) FR1 comprisingamino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii)FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem, and comprising (a) a VH domain comprising an amino acid sequenceof SEQ ID NO:11 with up to 15 amino acid substitutions; and (b) avariable light chain domain comprising an amino acid sequence of SEQ IDNO:12 with up to 15 amino acid substitutions.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1 beta, comprising a VH sequence of SEQ ID NO:11and a VL sequence of SEQ ID NO:12.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a heavy chain amino acid sequenceof SEQ ID NO:20 and a light chain amino acid sequence of SEQ ID NO:19.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, comprising a heavy chain amino acid sequenceof SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:19.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, (d) ahuman heavy chain framework with (i) FR1 comprising amino acid residuesE2, G26, V28, and K30, (ii) FR3 comprising amino acid residues R66, R83,and K94, and a VL domain comprising (e) CDR-L1 comprising the amino acidsequence of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence of SEQ IDNO:8, and (h) a human light chain framework with (i) FR1 comprisingamino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii)FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem, wherein an antibody Fab fragment of the antibody binds (i) tohuman VEGF121 with a K_(D) of less than 10 pM as measured by surfaceplasmon resonance, and (ii) to human IL-1beta with a K_(D) of less than30 pM as measured by surface plasmon resonance.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:11;and (b) a VL domain comprising an amino acid sequence having at least90% sequence identity to the amino acid sequence of SEQ ID NO:12;wherein an antibody Fab fragment of the antibody binds (i) to humanVEGF121 with a K_(D) of less than 10 pM as measured by surface plasmonresonance, and (ii) to human IL-1beta with a K_(D) of less than 30 pM asmeasured by surface plasmon resonance.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:11;and (b) a VL domain comprising an amino acid sequence having at least90% sequence identity to the amino acid sequence of SEQ ID NO:12;wherein an antibody Fab fragment of the antibody exhibits an aggregationonset temperature of more than 70° C.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and a(VL domain) comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:11;and (b) a VL domain comprising an amino acid sequence having at least90% sequence identity to the amino acid sequence of SEQ ID NO:12;wherein an antibody Fab fragment of the antibody exhibits a meltingtemperature of more than 80° C. as measured by dynamic light scattering.

In one aspect the invention provides an antibody that binds to humanVEGF and to human IL-1beta, wherein the antibody comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:11;and (b) a VL domain comprising an amino acid sequence having at least90% sequence identity to the amino acid sequence of SEQ ID NO:12; andwherein binding of an antibody Fab fragment of the antibody to humanIL-1beta inhibits binding of IL-1beta to IL-1betaR1 with an IC50 of lessthan 30 nM as measured by surface plasmon resonance.

One embodiment of the invention relates to an antibody fragment thatbinds to human VEGF and to human IL-1beta. One embodiment of theinvention relates to a bispecific antibody fragment that binds to humanVEGF and to human IL-1beta. In one embodiment the antibody fragment isselected from Fv, Fab, Fab′, Fab′-SH, F(ab′)₂ or single chain antibodiesderived therefrom. One embodiment of the invention relates to a Fabfragment that binds to human VEGF and to human IL-1beta. One embodimentof the invention relates to an Fv fragment that binds to human VEGF andto human IL-1beta.

One embodiment of the invention relates to a full length IgG antibodythat binds to human VEGF and to human IL-1beta.

In one aspect the invention provides an isolated nucleic acid encodingthe antibody of the invention.

In one aspect the invention provides a host cell comprising the nucleicacid of the invention.

In one aspect the invention provides an expression vector comprising thenucleic acid of the invention.

In one aspect the invention provides a method of producing an antibodythat binds to human VEGF and to human IL-1beta comprising culturing thehost cell of the invention so that the antibody is produced.

In one aspect the invention provides the antibody produced by the methodof the invention.

In one aspect the invention provides a pharmaceutical formulationcomprising the antibody of the invention and a pharmaceuticallyacceptable carrier.

In one aspect the invention provides the antibody of the invention foruse as a medicament, in one embodiment for use in the treatment of avascular disease.

In one aspect the invention provides the use of the antibody of theinvention or the pharmaceutical composition of the invention in themanufacture of a medicament, in one embodiment a medicament for thetreatment of a vascular disease.

In one aspect the invention provides a method of treating an individualhaving a vascular disease comprising administering to the individual aneffective amount of the antibody of the invention or the pharmaceuticalcomposition of the invention.

In one aspect the invention provides a method of inhibiting angiogenesisin an individual comprising administering to the individual an effectiveamount of the antibody of the invention or the pharmaceuticalcomposition of the invention to inhibit angiogenesis

According to the invention a therapeutic anti-VEGF/anti-IL-1betaantibody is provided that is capable of binding to its target antigenssimultaneously, even when provided as a bispecific Fab fragment. Inaddition the antibody of the invention provides several valuableproperties that allow its therapeutic application, like high affinity,hydrophilicity, and high stability. The antibody of the invention can beprovided in high concentrations liquid formulations with a viscositysuitable for ocular application. The antibody of the invention issuitable for the treatment of ocular vascular diseases.

DESCRIPTION OF THE FIGURES

FIG. 1 : Schematic illustration of the Fab fragment of ananti-VEGF/anti-IL-1beta antibody of the invention. Shown is a top downview of a cognate VH/VL pair including the arrangement of CDR amino acid(upper image). VH domain is indicated in grey, VL domain is indicated inwhite. Furthermore, the spatial arrangement of the CDR regions isindicated. Paratope regions of an antibody of the invention ishighlighted (lower image), with the VEGF paratope being arranged in theregions of H-CDR2, L-CDR1 and L-CDR2 and the IL-1beta paratope beingarranged in the regions of H-CDR1, H-CDR3 and L-CDR2.

FIG. 2 : Amino acid sequences of VH domains of examplaryanti-VEGF/anti-IL-1beta antibodies of the invention. Kabat numbering ofthe amino acid position is indicated, as well as the CDR and FR regions.Amino acid positions contributing to the VEGF paratope, as well as theIL-1beta paratope as identified in Example 8 are highlighted.

FIG. 3 : Amino acid sequences of VL domains of exemplaryanti-VEGF/anti-IL-1beta antibodies of the invention. Kabat numbering ofthe amino acid position is indicated, as well as the CDR and FR regions.Amino acid positions contributing to the VEGF paratope, as well as theIL-1beta paratope as identified in Example 8 are highlighted.

FIG. 4 : Simultaneous antigen binding of anti-VEGF/anti-IL-1betaantibody 1HVL12.85 to VEGF and IL-1beta as assessed via SPR according toExample 5.

FIG. 5 : Simultaneous antigen binding of anti-VEGF/anti-IL-1betaantibody RO7200394 to VEGF and IL-1beta as assessed via SPR according toExample 5.

FIG. 6 : Simultaneous antigen binding of prior artanti-VEGF/anti-IL-1beta antibody 0032 to VEGF and IL-1beta as assessedvia SPR according to Example 5.

FIG. 7A: Inhibition of binding of VEGF to hVEGFR2 in presence ofantibody RO7200394 (Fab fragment) as assessed in Example 6. Receptorbinding inhibition was assessed in presence and absence of the othertarget antigen of the bispecific antibody, IL-1beta.

FIG. 7B: Inhibition of binding of VEGF to hVEGFR2 in presence of priorart antibody 0032 (full length IgG) as assessed in Example 6. Receptorbinding inhibition was assessed in presence and absence of the othertarget antigen of the bispecific antibody, IL-1beta.

FIG. 8A: Inhibition of binding of IL-1beta to IL-1betaR1 in presence ofantibody RO7200394 (Fab fragment) as assessed in Example 6. Receptorbinding inhibition was assessed in presence and absence of the othertarget antigen of the bispecific antibody, VEGF.

FIG. 8B: Inhibition of binding of IL-1beta to IL-1betaR1 in presence ofprior art antibody 0032 (full length IgG) as assessed in Example 6.Receptor binding inhibition was assessed in presence and absence of theother target antigen of the bispecific antibody, VEGF.

FIG. 9 : Competition ELISA assessing VEGF121-binding to VEGF-R1 inpresence of indicated antibodies as assessed in Example 6

FIG. 10 : Competition ELISA assessing VEGF165-binding to VEGF-R1 inpresence of indicated antibodies as assessed in Example 6

FIG. 11 : Results of Hydrophobic Interaction Chromatography (HIC) ofantibodies of invention as assessed in Example 7

FIG. 12 : Results of Hydrophobic Interaction Chromatography (HIC) ofprior art antibody 0032 (Example 7)

FIG. 13 : Viscosity of antibody 1HVL12.85 as assessed in Example 8

FIG. 14 : Viscosity of antibody R07200394 as assessed in Example 8

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular, and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

Unless otherwise defined herein the term “comprising of” shall includethe term “consisting of”.

The term “about” as used herein in connection with a specific value(e.g. temperature, concentration, time and others) shall refer to avariation of +/−1% of the specific value that the term “about” refersto.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desired antigen-bindingactivity.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC) methods. For a review of methods for assessment of antibodypurity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is ofthe IgG1 isotype. In certain embodiments, the antibody is of the IgG1isotype with the P329G, L234A and L235A mutation to reduce Fc-regioneffector function. In other embodiments, the antibody is of the IgG2isotype. In certain embodiments, the antibody is of the IgG4 isotypewith the S228P mutation in the hinge region to improve stability of IgG4antibody. The heavy chain constant domains that correspond to thedifferent classes of immunoglobulins are called α, δ, ε, γ, and μ,respectively. The light chain of an antibody may be assigned to one oftwo types, called kappa (κ) and lambda (λ), based on the amino acidsequence of its constant domain.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. Unless otherwise specified herein, numbering of amino acidresidues in the Fc region or constant region is according to the EUnumbering system, also called the EU index, as described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, M D, 1991.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1 q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindtet al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).In the antibody of the invention, a single pair of a VH domain and a VLdomain, i.e. a cognate VH/VL pair, specifically binds to its twotargets: VEGF and IL-1beta.

A “DutaFab” is a bispecific antibody as disclosed in WO2012/163520. In aDutaFab a single pair of a VH domain and a VL domain specifically bindsto two different epitopes, wherein one paratope comprises amino acidresidues from from CDR-H2, CDR-L1 and CDR-L3 and the other paratopecomprises amino residues from CDR-H1, CDR-H3 and CDR-L2. DutaFabscomprise two non-overlapping paratopes within a cognate VH/VL pair andmay simultaneously bind to the two different epitopes. DutaFabs andmethods for their generation by screening of libraries comprisingmonospecific Fab fragments are disclosed in WO2012/163520.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda M D (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

A “paratope” or “antigen binding site”, as used interchangeably herein,refers to a part of an antibody which recognizes and binds to anantigen. A paratope is formed by several individual amino acid residuesfrom the antibody's heavy and light chain variable domains arranged thatare arranged in spatial proximity in the tertiary structure of the Fvregion. The antibodies of the invention comprise two “non-overlapping”paratopes in one cognate VH/VL pair. By “non-overlapping” is meant thatnone of the amino acids that are comprised in one of the two paratopesis comprised in the other paratope.

As used herein a “VEGF paratope” is a paratope or antigen binding sitethat binds to VEGF. The VEGF paratope of an antibody of the inventioncomprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of theantibody.

As used herein an “IL-1beta paratope” is a paratope or antigen bindingsite that binds to IL-1beta. The IL-1beta paratope of an antibody of theinvention comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2of the antibody.

The term “VEGF”, as used herein, refers to any native VEGF from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length”, unprocessed VEGF as well as any form of VEGFthat results from processing in the cell. The term also encompassesnaturally occurring variants of VEGF, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human VEGF is shown inSEQ ID NO:26.

The terms “anti-VEGF antibody” and “an antibody that binds to VEGF”refer to an antibody that is capable of binding VEGF with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting VEGF. In one embodiment, the extent ofbinding of an anti-VEGF antibody to an unrelated, non-VEGF protein isless than about 10% of the binding of the antibody to VEGF as measured,e.g., by surface plasmon resonance (SPR). In certain embodiments, anantibody that binds to VEGF has a dissociation constant (K_(D)) of ≤1nM, ≤0.1 nM, or ≤0.01 nM. An antibody is said to “specifically bind” toVEGF when the antibody has a K_(D) of 1 μM or less.

The term “IL-1beta”, as used herein, refers to any native IL-1beta fromany vertebrate source, including mammals such as primates (e.g. humans)and rodents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length”, unprocessed IL-1beta as well as any form ofIL-1beta that results from processing in the cell. The term alsoencompasses naturally occurring variants of IL-1beta, e.g., splicevariants or allelic variants. The amino acid sequence of an exemplaryhuman IL-1beta is shown in SEQ ID NO:27.

The terms “anti-IL-1beta antibody” and “an antibody that binds toanti-IL-1beta” refer to an antibody that is capable of bindinganti-IL-1beta with sufficient affinity such that the antibody is usefulas a diagnostic and/or therapeutic agent in targeting anti-IL-1beta. Inone embodiment, the extent of binding of an anti-anti-IL-1beta antibodyto an unrelated, non-anti-IL-1beta protein is less than about 10% of thebinding of the antibody to anti-IL-1beta as measured, e.g., by surfaceplasmon resonance (SPR). In certain embodiments, an antibody that bindsto IL-1beta has a dissociation constant (K_(D)) of ≤1 nM, ≤0.1 nM, or≤0.03 nM. An antibody is said to “specifically bind” to anti-IL-1betawhen the antibody has a K_(D) of 1μM or less.

An antibody of the invention “simultaneously binds to human VEGF andhuman IL-1beta”, which means that (a) an antibody Fab fragment of theinvention that is bound to human IL-1beta (also) specifically binds tohuman VEGF, and (b) an antibody Fab fragment of the invention that isbound to human VEGF (also) specifically binds to human IL-1beta.Simultaneous binding may be assessed with methods known in the art, e.g.by surface plasmon resonance as described herein.

The term “complementarity determining regions” or “CDRs” as used hereinrefers to each of the regions of an antibody variable domain which arehypervariable in sequence and contain antigen-contacting residues.Generally, antibodies comprise six CDRs: three in the VH domain (CDR-H1,CDR-H2, CDR-H3), and three in the VL domain (CDR-L1, CDR-L2, CDR-L3).Unless otherwise indicated, CDR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according to theKabat numbering system (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, M D, 1991).

“Framework” or “FR” as used herein refers to variable domain amino acidresidues other than CDR residues. The framework of a variable domaingenerally consists of four framework domains: FR1, FR2, FR3, and FR4.Accordingly, the CDR and FR amino acid sequences generally appear in thefollowing sequence in the (a) VH domain:FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; and (b) in the VL domain:FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4.

According to the Kabat numbering system, as is used herein, frameworkand CDR regions are located at the following regions of the variabledomains:

FR1 CDR-1 FR2 CDR2 FR3 CDR3 FR4 VH 1-30 31-35b* 36-49 50-65 66-94 95-102103-113 VL 1-23 24-34 35-49 50-56 57-88 89-97  98-107 *in CDR-H1additional amino acids between position 35b and 36 may be present,herein referred to as positions “35c”, “35d” and “35e” as illustrated inFigure 2

The amino acid positions according to the Kabat numbering systemreferred to herein are illustrated in FIG. 2 in an alignment with theamino acid sequences of antibodies of the invention. References to aminoacids at a certain position within the amino acid sequence are hereinmade as well known in the art by stating the respective amino acid andthe amino acid position, e.g. “E2” refers to a glutamic acid residuelocated at Kabat position 2 of the amino acid sequence of the respectiveantibody domain.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)). Affinity can be measured by common methods known inthe art, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are describedherein.

The term “epitope” denotes the site on an antigen, either proteinaceousor non-proteinaceous, to which an antibody binds. Epitopes can be formedboth from contiguous amino acid stretches (linear epitope) or comprisenon-contiguous amino acids (conformational epitope), e.g. coming inspatial proximity due to the folding of the antigen, i.e. by thetertiary folding of a proteinaceous antigen. Linear epitopes aretypically still bound by an antibody after exposure of the proteinaceousantigen to denaturing agents, whereas conformational epitopes aretypically destroyed upon treatment with denaturing agents. An epitopecomprises at least 3, at least 4, at least 5, at least 6, at least 7, or8-10 amino acids in a unique spatial conformation.

Screening for antibodies binding to a particular epitope (i.e., thosebinding to the same epitope) can be done using methods routine in theart such as, e.g., without limitation, alanine scanning, peptide blots(see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis,epitope excision, epitope extraction, chemical modification of antigens(see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

Antigen Structure-based Antibody Profiling (ASAP), also known asModification-Assisted Profiling (MAP), allows to bin a multitude ofmonoclonal antibodies specifically binding to VEGF or IL-1beta based onthe binding profile of each of the antibodies from the multitude tochemically or enzymatically modified antigen surfaces (see, e.g., US2004/0101920). The antibodies in each bin bind to the same epitope whichmay be a unique epitope either distinctly different from or partiallyoverlapping with epitope represented by another bin.

Also competitive binding can be used to easily determine whether anantibody binds to the same epitope of VEGF or IL-1beta as, or competesfor binding with, a reference antibody of the invention. For example, an“antibody that binds to the same epitopes on VEGF and IL-1beta” as areference-antibody refers to an antibody that blocks binding of thereference-antibody to its antigens in respective competition assays by50% or more, and conversely, the reference antibody blocks binding ofthe antibody to its antigen in respective competition assays by 50% ormore. Also for example, to determine if an antibody binds to the sameepitope as a reference-antibody, the reference-antibody is allowed tobind to VEGF or IL-1beta under saturating conditions. After removal ofthe excess of the reference-antibody, the ability of an antibody inquestion to bind to VEGF or IL-1beta is assessed. If the antibody inquestion is able to bind to VEGF or IL-1beta after saturation binding ofthe reference-antibody, it can be concluded that the antibody inquestion binds to a different epitope than the reference-antibody. But,if the antibody in question is not able to bind to VEGF or IL-1betaafter saturation binding of the reference-antibody, then the antibody inquestion may bind to the same epitope as the epitope bound by thereference-antibody. To confirm whether the antibody in question binds tothe same epitope or is just hampered from binding by steric reasonsroutine experimentation can be used (e.g., peptide mutation and bindinganalyses using ELISA, RIA, surface plasmon resonance, flow cytometry orany other quantitative or qualitative antibody-binding assay availablein the art). This assay should be carried out in two set-ups, i.e. withboth of the antibodies being the saturating antibody. If, in bothset-ups, only the first (saturating) antibody is capable of binding toVEGF or IL-1beta, then it can be concluded that the antibody in questionand the reference-antibody compete for binding to VEGF or IL-1beta.

In some embodiments two antibodies are deemed to bind to the same or anoverlapping epitope if a 1-, 5-, 10-, 20- or 100-fold excess of oneantibody inhibits binding of the other by at least 50%, at least 75%, atleast 90% or even 99% or more as measured in a competitive binding assay(see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

In some embodiments two antibodies are deemed to bind to the sameepitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody also reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity for the purposes of the alignment. Alignment forpurposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA programpackage. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.Alternatively, the percent identity values can be generated using thesequence comparison computer program ALIGN-2. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc., and thesource code has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087 and is described in WO 2000/005319.

Unless otherwise indicated, For for purposes herein, however, % aminoacid sequence identity values are generated using the ggsearch programof the FASTA package version 36.3.8c or later with a BLOSUM50 comparisonmatrix. The FASTA program package was authored by W. R. Pearson and D.J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”,PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequencecomparison” Meth. Enzymol. 266:227-258; and Pearson et. al. (1997)Genomics 46:24-36 and is publicly available fromwww.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server accessible atfasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare thesequences, using the ggsearch (global protein:protein) program anddefault options (BLOSUM50; open: −10; ext: −2; Ktup=2) to ensure aglobal, rather than local, alignment is performed. Percent amino acididentity is given in the output alignment header.

The term “nucleic acid molecule” or “polynucleotide” includes anycompound and/or substance that comprises a polymer of nucleotides. Eachnucleotide is composed of a base, specifically a purine- or pyrimidinebase (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil(U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.Often, the nucleic acid molecule is described by the sequence of bases,whereby said bases represent the primary structure (linear structure) ofa nucleic acid molecule. The sequence of bases is typically representedfrom 5′ to 3′. Herein, the term nucleic acid molecule encompassesdeoxyribonucleic acid (DNA) including e.g. complementary DNA (cDNA) andgenomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA),synthetic forms of DNA or RNA, and mixed polymers comprising two or moreof these molecules. The nucleic acid molecule may be linear or circular.In addition, the term nucleic acid molecule includes both, sense andantisense strands, as well as single stranded and double stranded forms.Moreover, the herein described nucleic acid molecule can containnaturally occurring or non-naturally occurring nucleotides. Examples ofnon-naturally occurring nucleotides include modified nucleotide baseswith derivatized sugars or phosphate backbone linkages or chemicallymodified residues. Nucleic acid molecules also encompass DNA and RNAmolecules which are suitable as a vector for direct expression of anantibody of the invention in vitro and/or in vivo, e.g. in a host orpatient. Such DNA (e.g. cDNA) or RNA (e.g. mRNA) vectors, can beunmodified or modified. For example, mRNA can be chemically modified toenhance the stability of the RNA vector and/or expression of the encodedmolecule so that mRNA can be injected into a subject to generate theantibody in vivo (see e.g. Stadler ert al, Nature Medicine 2017,published online 12 Jun. 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding” an antibody refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells”, which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

The term “pharmaceutical composition” or “pharmaceutical formulation”refers to a preparation which is in such form as to permit thebiological activity of an active ingredient contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the pharmaceutical compositionwould be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition or formulation, other than an activeingredient, which is nontoxic to a subject. A pharmaceuticallyacceptable carrier includes, but is not limited to, a buffer, excipient,stabilizer, or preservative.

An “effective amount” of an agent, e.g., a pharmaceutical composition,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “ocular disease,” as used herein, includes any ocular diseaseassociated with pathological angiogenesis and/or atrophy. An oculardisease may be characterized by altered or unregulated proliferationand/or invasion of new blood vessels into the structures of oculartissues such as the retina or cornea. An ocular disease may becharacterized by atrophy of retinal tissue (photoreceptors and theunderlying retinal pigment epithelium (RPE) and choriocapillaris).Non-limiting ocular diseases include, for example, AMD (e.g., wet AMD,dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)),macular degeneration, macular edema, DME (e.g., focal, non-center DMEand diffuse, center-involved DME), retinopathy, diabetic retinopathy(DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), andhigh-altitude DR), other ischemia-related retinopathies, ROP, retinalvein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms),CNV (e.g., myopic CNV), corneal neovascularization, diseases associatedwith corneal neovascularization, retinal neovascularization, diseasesassociated with retinal/choroidal neovascularization, central serousretinopathy (CSR), pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, FEVR, Coats' disease, Norrie Disease, retinalabnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG),subconjunctival hemorrhage, rubeosis, ocular neovascular disease,neovascular glaucoma, retinitis pigmentosa (RP), hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, cystoid macular edema (CME), vasculitis, papilloedema,retinitis, including but not limited to CMV retinitis, ocular melanoma,retinal blastoma, conjunctivitis (e.g., infectious conjunctivitis andnon-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis (also known as Leber's congenital amaurosis or LCA), uveitis(including infectious and non-infectious uveitis), choroiditis (e.g.,multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye,traumatic eye injury, Sjögren's disease, and other ophthalmic diseaseswherein the disease or disease is associated with ocularneovascularization, vascular leakage, and/or retinal edema or retinalatrophy. Additional exemplary ocular diseases include retinoschisis(abnormal splitting of the retina neurosensory layers), diseasesassociated with rubeosis (neovascularization of the angle) and diseasescaused by the abnormal proliferation of fibrovascular or fibrous tissue,including all forms of proliferative vitreoretinopathy. Exemplarydiseases associated with comeal neovascularization include, but are notlimited to, epidemic keratoconjunctivitis, vitamin A deficiency, contactlens overwear, atopic keratitis, superior limbic keratitis, terygiumkeratitis sicca, Sjögren's syndrome, acne rosacea, phylectenulosis,syphilis, Mycobacteria infections, lipid degeneration, chemical burns,bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpeszoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis,scleritis, Stevens-Johnson syndrome, periphigoid radial keratotomy, andcorneal graph rejection. Exemplary diseases associated with choroidalneovascularization and defects in the retina vasculature, includingincreased vascular leak, aneurisms and capillary drop-out include, butare not limited to, diabetic retinopathy, macular degeneration, sicklecell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget'sdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, retina edema(including macular edema), Eales disease, Behcet's disease, infectionscausing retinitis or choroiditis (e.g., multifocal choroidits), presumedocular histoplasmosis, Best's disease (vitelliform maculardegeneration), myopia, optic pits, pars planitis, retinal detachment(e.g., chronic retinal detachment), hyperviscosity syndromes,toxoplasmosis, trauma, and post-laser complications. Exemplary diseasesassociated with atrophy of retinal tissues (photoreceptors and theunderlying RPE) include, but are not limited to, atrophic ornonexudative AMD (e.g., geographic atrophy or advanced dry AMD), macularatrophy (e.g., atrophy associated with neovascularization and/orgeographic atrophy), diabetic retinopathy, Stargardt's disease, SorsbyFundus Dystrophy, retinoschisis and retinitis pigmentosa.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

2. Detailed Description of the Embodiments of the Invention

In one aspect, the invention is based, in part, on the provision ofbispecific antibodies for therapeutic application. In certain aspects,antibodies that bind to human VEGF and human IL-1beta are provided.Antibodies of the invention are useful, e.g., for the diagnosis ortreatment of vascular diseases, e.g. ocular vascular diseases.

A. Exemplary Antibodies that Bind to Human VEGF and Human IL-1Beta

In one aspect, the invention provides antibodies that bind to human VEGFand human IL-1beta. In one aspect, provided are isolated antibodies thatbind to human VEGF and human IL-1beta. In one aspect, the inventionprovides antibodies that specifically bind to human VEGF and humanIL-1beta.

In certain aspects, an antibody that binds to human VEGF and to humanIL-1beta is provided, wherein the antibody comprises a VEGF paratope(i.e. an antigen binding site that binds to VEGF) and an IL-1betaparatope (i.e. an antigen binding site that binds to IL-1beta) withinone cognate pair of a VL domain and a VH domain, wherein

-   -   the VEGF paratope comprises amino acid residues from CDR-H2,        CDR-L1 and CDR-L3 of the antibody, wherein the IL-1 beta        paratope comprises amino acid residues from the CDR-H1, CDR-H3        and CDR-L2 of the antibody; and/or    -   the pair of the variable light chain domain and the variable        heavy chain domain simultaneously binds to human VEGF and human        IL-1beta; and/or    -   none of the amino acids that are comprised in the VEGF paratope        are comprised in the IL-1beta paratope; and/or    -   none of the amino acids that are comprised in the IL-1beta        paratope are comprised in the VEGF paratope; and/or    -   the antibody binds to the same epitope on human VEGF and to the        same epitope on human IL-1beta as an antibody with a variable        heavy chain domain of SEQ ID NO: 11 and a variable light chain        domain of SEQ ID NO: 12; and/or    -   an antibody Fab fragment of the antibody binds (i) to human        VEGF121 with a K_(D) of less than 10 pM as measured by surface        plasmon resonance, and (ii) to human IL-1beta with a K_(D) of        less than 30 pM as measured by surface plasmon resonance; and/or    -   an antibody Fab fragment of the antibody exhibits an aggregation        onset temperature of more than 70° C.; and/or    -   an antibody Fab fragment of the antibody exhibits a melting        temperature of more than 80° C. as measured by dynamic light        scattering; and/or    -   binding of an antibody Fab fragment of the antibody to human        VEGF inhibits binding of VEGF to VEGFR2 with an IC50 of less        than 50 nM as measured by surface plasmon resonance; and wherein        binding of an antibody Fab fragment of the antibody to human        IL-1beta inhibits binding of IL-1beta to IL-1betaR1 with an IC50        of less than 30 nM as measured by surface plasmon resonance.

In another aspect, the invention provides an antibody comprising a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8.

In another aspect, the invention provides an antibody comprising a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, (d) ahuman heavy chain framework with (i) FR1 comprising amino acid residuesE2, G26, V28, and K30, (ii) FR3 comprising amino acid residues R66, R83,and K94: and a VL domain comprising (e) CDR-L1 comprising the amino acidsequence of SEQ ID NO:16, (0 CDR-L2 comprising the amino acid sequenceof SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence of SEQ IDNO:8, and (h) a human light chain framework with (i) FR1 comprisingamino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii)FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem

In another aspect, the invention provides an antibody comprising a VHdomain comprising amino acid residues E2, G26, V28, K30, W31, N35b,D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96,F98, and D101, and a VL domain comprising amino acid residues I2, Y27,W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91,R92, Y93, H94, and Y96, wherein the numbering of the VH and VL domainsis according to the Kabat numbering system. In one embodiment theantibody comprises a VEGF paratope comprising the following amino acidresidues in the VH domain: D55, H56, Y58, T61, K62, F63, I64, R66, andR83, and the following amino acid residues in the VL domain: 12, Y27,W27a, S27c, S27d, E67, D68, Q69, R92, Y93, H94, and Y96; and an IL-1betaparatope comprising the following amino acid residues in the VH domain:E2, G26, V28, K30, W31, N35b, D35c, K52a, K94, D95, V96, F98, and D101,and the following amino acid residues in the VL domain: L32, Y49, D50,Y53, K54, L56, G57, Y91.

In another aspect, the invention provides an antibody comprising (a) aVH domain comprising an amino acid sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequence of SEQ ID NO:11; and (b) aVL domain comprising an amino acid sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequence of SEQ ID NO:12.

In another aspect, the invention provides an antibody comprising (a) aVH domain comprising an amino acid sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to the amino acid sequence of SEQ ID NO:11, whereinthe VH domain comprises amino acid residues E2, G26, V28, K30, W31,N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95,V96, F98, and D101; and (b) a VL domain comprising an amino acidsequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acidsequence of SEQ ID NO:12, wherein the VL domain comprises amino acidresidues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57,E67, D68, Q69, Y91, R92, Y93, H94, and Y96, wherein the numbering of theVH and VL domains is according to the Kabat numbering system.

In another aspect, the invention provides an antibody comprising a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:11;and (b) a VL domain comprising an amino acid sequence having at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to the amino acid sequence of SEQ ID NO:12.

In another aspect, the invention provides an antibody comprising a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:11,wherein the VH domain comprises amino acid residues E2, G26, V28, K30,R66, R83, and K94; and (b) a VL domain comprising an amino acid sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQID NO:12, wherein the VL domain comprises amino acid residues I2, Y49,G57, E67, D68, and Q69, wherein the numbering of the VH and VL domainsis according to the Kabat numbering system.

In another aspect, the invention provides an antibody comprising a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, (d) ahuman heavy chain framework with (i) FR1 comprising amino acid residuesE2, G26, V28, and K30, (ii) FR3 comprising amino acid residues R66, R83,and K94, and a VL domain comprising (e) CDR-L1 comprising the amino acidsequence of SEQ ID NO:16, (0 CDR-L2 comprising the amino acid sequenceof SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence of SEQ IDNO:8, and (h) a human light chain framework with (i) FR1 comprisingamino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii)FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem, comprising (a) a VH domain comprising an amino acid sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQID NO:11; and (b) a VL domain comprising an amino acid sequence havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to the amino acid sequence of SEQ IDNO:12.

In another aspect, the invention provides an antibody comprising (a) aVH domain comprising an amino acid sequence of SEQ ID NO:11 with 1 to15, 1 to 10, or 1 to 5 amino acid substitutions; and (b) a variablelight chain domain comprising an amino acid sequence of SEQ ID NO:12with 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

In another aspect, the invention provides an antibody comprising (a) aVH domain comprising an amino acid sequence of SEQ ID NO:11 with 1 to15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acidsubstitutions are located at positions 3 to 25, 36 to 49, 97 to 82c, 84to 93, or 103 to 113 of SEQ ID NO:11; and (b) a variable light chaindomain comprising an amino acid sequence of SEQ ID NO:12 with 1 to 15, 1to 10, or 1 to 5 amino acid substitutions, wherein the amino acidsubstitutions are located at positions 1, 4, 6, 8 to 23, 35 to 48, 58 to66, 70 to 88, or 98 to 107 of SEQ ID NO:12, wherein the numbering of theVH and VL domains is according to the Kabat numbering system.

In another aspect, the invention provides an antibody comprising a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and aVL domain comprising (d) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,comprising (a) a VH domain comprising an amino acid sequence of SEQ IDNO:11 with 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions; and (b)a variable light chain domain comprising an amino acid sequence of SEQID NO:12 with 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

In another aspect, the invention provides an antibody comprising a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, (d) ahuman heavy chain framework with (i) FR1 comprising amino acid residuesE2, G26, V28, and K30, (ii) FR3 comprising amino acid residues R66, R83,and K94; and a VL domain comprising (e) CDR-L1 comprising the amino acidsequence of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence of SEQ IDNO:8, and (h) a human light chain framework with (i) FR1 comprisingamino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii)FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein thenumbering of the VH and VL domains is according to the Kabat numberingsystem, and comprising (a) a VH domain comprising an amino acid sequenceof SEQ ID NO:11 with 1 to 15, 1 to 10, or 1 to 5 amino acidsubstitutions; and (b) a variable light chain domain comprising an aminoacid sequence of SEQ ID NO:12 with 1 to 15, 1 to 10, or 1 to 5 aminoacid substitutions.

In one aspect, the invention provides an antibody comprising a VH domainhaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO:11. Incertain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an antibody that binds to human VEGF and humanIL-1beta comprising that sequence retains the ability to bind to tohuman VEGF and human IL-1beta. In certain aspects, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:11. In certain aspects, substitutions, insertions, or deletions occurin regions outside the CDRs (i.e., in the FRs). In a particular aspect,the VH comprises (a) CDR-H1 comprising the amino acid sequence of SEQ IDNO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14,and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15.

In one aspect, the invention provides an antibody comprising a VL domainhaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO:12. Incertain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an antibody that binds to human VEGF and humanIL-1beta comprising that sequence retains the ability to bind to tohuman VEGF and human IL-1beta. In certain aspects, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQ IDNO:12. In certain aspects, substitutions, insertions, or deletions occurin regions outside the CDRs (i.e., in the FRs). In a particular aspect,the VL comprises (d) CDR-L1 comprising the amino acid sequence of SEQ IDNO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:17,and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8.

In another aspect, an antibody that binds to human VEGF and humanIL-1beta is provided, wherein the antibody comprises a VH sequence as inany of the aspects provided above, and a VL sequence as in any of theaspects provided above. In one aspect, the antibody comprises the VH andVL sequences in SEQ ID NO:11 and SEQ ID NO:12, respectively, includingpost-translational modifications of those sequences.

In another aspect, an antibody that binds to human VEGF and humanIL-1beta is provided, wherein the antibody comprises a heavy chain aminoacid sequence of SEQ ID NO:20 and a light chain amino acid sequence ofSEQ ID NO:19.

In another aspect, an antibody that binds to human VEGF and humanIL-1beta is provided, wherein the antibody comprises a heavy chain aminoacid sequence of SEQ ID NO:18 and a light chain amino acid sequence ofSEQ ID NO:19.

In a further aspect of the invention, an antibody that binds to humanVEGF and human IL-1beta according to any of the above aspects is amonoclonal antibody. In one aspect, an antibody that binds to human VEGFand human IL-1beta is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv,diabody, or F(ab′)₂ fragment. In another aspect, the antibody is a fulllength antibody.

In a further aspect, an antibody that binds to human VEGF and humanIL-1beta according to any of the above aspects may incorporate any ofthe features, singly or in combination, as described in Sections 1-7below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein binds to VEGF with adissociation constant (K_(D)) of ≤1 nM, ≤0.1 nM, or ≤0.01 nM. In certainembodiments, an antibody that binds to IL-1beta has a dissociationconstant (K_(D)) of ≤1 nM, ≤0.1 nM, or ≤0.03 nM.

In one aspect, K_(D) is measured using a BIACORE® surface plasmonresonance assay.

For example, the K_(D) of antibody binding to VEGF is measured in anassay using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc.,Piscataway, NJ) performed at 25° C. with immobilized VEGF121 on C1 chipsat ˜10 response units (RU). For kinetics measurements, two-fold serialdilutions of Fab (1.2-100 nM) are injected in HBS-P+ (10 mM HEPES, 150mM NaCl pH 7.4, 0.05% Surfactant P20) at 25° C. at a flow rate ofapproximately 30 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999).

For example, the K_(D) of antibody binding to IL-1beta is measured in anassay using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc.,Piscataway, NJ) performed at 25° C. with immobilized bispecific antibodyon C1 chips at ˜20 response units (RU). For kinetics measurements,two-fold serial dilutions of human IL-1beta (0.74 to 60 nM) are injectedin HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) at 25°C. at a flow rate of approximately 30 μl/min. Association rates (k_(om))and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIACORE® Evaluation Software version3.2) by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) is calculatedas the ratio k_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999).

2. Antibody Fragments

In certain aspects, an antibody provided herein is an antibody fragment.

In one aspect, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2fragment, in particular a Fab fragment. Papain digestion of intactantibodies produces two identical antigen-binding fragments, called“Fab” fragments containing each the heavy- and light-chain variabledomains (VH and VL, respectively) and also the constant domain of thelight chain (CL) and the first constant domain of the heavy chain (CH1).The term “Fab fragment” thus refers to an antibody fragment comprising alight chain comprising a VL domain and a CL domain, and a heavy chainfragment comprising a VH domain and a CH1 domain. “Fab′ fragments”differ from Fab fragments by the addition of residues at the carboxyterminus of the CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH are Fab′ fragments in which the cysteineresidue(s) of the constant domains bear a free thiol group. Pepsintreatment yields an F(ab′)2 fragment that has two antigen-binding sites(two Fab fragments) and a part of the Fc region. For discussion of Faband F(ab′)₂ fragments comprising salvage receptor binding epitoperesidues and having increased in vivo half-life, see U.S. Pat. No.5,869,046.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asrecombinant production by recombinant host cells (e.g., E. coli, CHO),as described herein.

3. Thermal Stability

Antibodies provided herein exhibite superior thermal stability. Incertain embodiments, a Fab fragment of an antibody provided hereinexhibits an aggregation onset temperature of more than 70° C. In certainembodiments, a Fab fragment of an antibody provided herein exhibits amelting temperature of more than 80° C. as measured by dynamic lightscattering.

4. Library-Derived Antibodies

In certain aspects, an antibody provided herein is derived from alibrary. Antibodies of the invention may be isolated by screeningcombinatorial libraries for antibodies with the desired activity oractivities. Methods for screening combinatorial libraries are reviewed,e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example,a variety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, e.g., inFrenzel et al. in mAbs 8:1177-1194 (2016); Bazan et al. in HumanVaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. inCritical Reviews in Biotechnology 36:276-289 (2016) as well as inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N J, 2001) and in Marks and Bradbury inMethods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa,N J, 2003).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. in Annual Review ofImmunology 12: 433-455 (1994). Phage typically display antibodyfragments, either as single-chain Fv (scFv) fragments or as Fabfragments. Libraries from immunized sources provide high-affinityantibodies to the immunogen without the requirement of constructinghybridomas. Alternatively, the naive repertoire can be cloned (e.g.,from human) to provide a single source of antibodies to a wide range ofnon-self and also self antigens without any immunization as described byGriffiths et al. in EMBO Journal 12: 725-734 (1993). Furthermore, naivelibraries can also be made synthetically by cloning unrearranged V-genesegments from stem cells, and using PCR primers containing randomsequence to encode the highly variable CDR3 regions and to accomplishrearrangement in vitro, as described by Hoogenboom and Winter in Journalof Molecular Biology 227: 381-388 (1992). Patent publications describinghuman antibody phage libraries include, for example: U.S. Pat. Nos.5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US PatentPublication Nos. 2005/0079574, 2007/0117126, 2007/0237764 and2007/0292936.

Further examples of methods known in the art for screening combinatoriallibraries for antibodies with a desired activity or activities includeribosome and mRNA display, as well as methods for antibody display andselection on bacteria, mammalian cells, insect cells or yeast cells.Methods for yeast surface display are reviewed, e.g., in Scholler et al.in Methods in Molecular Biology 503:135-56 (2012) and in Cherf et al. inMethods in Molecular biology 1319:155-175 (2015) as well as in Zhao etal. in Methods in Molecular Biology 889:73-84 (2012). Methods forribosome display are described, e.g., in He et al. in Nucleic AcidsResearch 25:5132-5134 (1997) and in Hanes et al. in PNAS 94:4937-4942(1997).

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

5. Multispecific Antibodies

In certain aspects, an antibody provided herein is a multispecificantibody. “Multispecific antibodies” are monoclonal antibodies that havebinding specificities for at least two different sites, i.e., differentepitopes on different antigens or different epitopes on the sameantigen. In certain aspects, the multispecific antibody has three ormore binding specificities.

Multispecific antibodies with three or more binding specificitiescomprising antibodies provided herein may be provided in an asymmetricform with a domain crossover in one or more binding arms of the sameantigen specificity, i.e. by exchanging the VH/VL domains (see e.g., WO2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, andKlein at al., MAbs 8 (2016) 1010-20). Various further molecular formatsfor multispecific antibodies are known in the art and are includedherein (see e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

6. Antibody Variants

In certain aspects, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toalter the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

In certain aspects, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the CDRs and FRs. Conservative substitutions areshown in the Table beolw under the heading of “preferred substitutions”.More substantial changes are provided in Table 1 under the heading of“exemplary substitutions”, and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for a member of another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore. CDR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g., bindingaffinity).

Alterations (e.g., substitutions) may be made in CDRs, e.g., to improveantibody affinity. Such alterations may be made in CDR “hotspots”, i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,NJ, (2001).) In some aspects of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves CDR-directed approaches, in which several CDR residues (e.g.,4-6 residues at a time) are randomized. CDR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain aspects, substitutions, insertions, or deletions may occurwithin one or more CDRs so long as such alterations do not substantiallyreduce the ability of the antibody to bind antigen. For example,conservative alterations (e.g., conservative substitutions as providedherein) that do not substantially reduce binding affinity may be made inthe CDRs. Such alterations may, for example, be outside of antigencontacting residues in the CDRs. In certain variant VH and VL sequencesprovided above, each CDR either is unaltered, or contains no more thanone, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex may beused to identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT (antibodydirected enzyme prodrug therapy)) or a polypeptide which increases theserum half-life of the antibody.

a) Glycosylation Variants

In certain aspects, an antibody provided herein is altered to increaseor decrease the extent to which the antibody is glycosylated. Additionor deletion of glycosylation sites to an antibody may be convenientlyaccomplished by altering the amino acid sequence such that one or moreglycosylation sites is created or removed.

Where the antibody comprises an Fc region, the oligosaccharide attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some aspects, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one aspect, antibody variants are provided having a non-fucosylatedoligosaccharide, i.e. an oligosaccharide structure that lacks fucoseattached (directly or indirectly) to an Fc region. Such non-fucosylatedoligosaccharide (also referred to as “afucosylated” oligosaccharide)particularly is an N-linked oligosaccharide which lacks a fucose residueattached to the first GlcNAc in the stem of the biantennaryoligosaccharide structure. In one aspect, antibody variants are providedhaving an increased proportion of non-fucosylated oligosaccharides inthe Fc region as compared to a native or parent antibody. For example,the proportion of non-fucosylated oligosaccharides may be at least about20%, at least about 40%, at least about 60%, at least about 80%, or evenabout 100% (i.e. no fucosylated oligosaccharides are present). Thepercentage of non-fucosylated oligosaccharides is the (average) amountof oligosaccharides lacking fucose residues, relative to the sum of alloligosaccharides attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2006/082515, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such antibodies having an increased proportion ofnon-fucosylated oligosaccharides in the Fc region may have improvedFcγRIIIa receptor binding and/or improved effector function, inparticular improved ADCC function. See, e.g., US 2003/0157108; US2004/0093621.

Examples of cell lines capable of producing antibodies with reducedfucosylation include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003/0157108; and WO 2004/056312, especially at Example 11), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng.87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006); and WO 2003/085107), or cells with reduced or abolished activityof a GDP-fucose synthesis or transporter protein (see, e.g.,US2004259150, US2005031613, US2004132140, US2004110282).

In a further aspect, antibody variants are provided with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region of the antibody is bisected by GlcNAc. Such antibodyvariants may have reduced fucosylation and/or improved ADCC function asdescribed above. Examples of such antibody variants are described, e.g.,in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al.,Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO2003/011878.

Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

b) Fc Region Variants

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG₁, IgG₂, IgG₃ or IgG₄ Fcregion) comprising an amino acid modification (e.g., a substitution) atone or more amino acid positions.

In certain aspects, the invention contemplates an antibody variant thatpossesses some but not all effector functions, which make it a desirablecandidate for applications in which the half life of the antibody invivo is important yet certain effector functions (such ascomplement-dependent cytotoxicity (CDC) and antibody-dependentcell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγR1, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.,Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad Sci. USA 95:652-656 (1998). C1q binding assays may alsobe carried out to confirm that the antibody is unable to bind Clq andhence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006); WO 2013/120929 A1).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain aspects, an antibody variant comprises an Fc region with oneor more amino acid substitutions which improve ADCC, e.g., substitutionsat positions 298, 333, and/or 334 of the Fc region (EU numbering ofresidues).

In certain aspects, an antibody variant comprises an Fc region with oneor more amino acid substitutions which diminish FcγR binding, e.g.,substitutions at positions 234 and 235 of the Fc region (EU numbering ofresidues). In one aspect, the substitutions are L234A and L235A (LALA).In certain aspects, the antibody variant further comprises D265A and/orP329G in an Fc region derived from a human IgG₁ Fc region. In oneaspect, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fcregion derived from a human IgG₁ Fc region. (See, e.g., WO 2012/130831).In another aspect, the substitutions are L234A, L235A and D265A(LALA-DA) in an Fc region derived from a human IgG₁ Fc region.

In some aspects, alterations are made in the Fc region that result inaltered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934(Hinton et al.). Those antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Such Fc variants include those with substitutions at one or more of Fcregion residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g.,substitution of Fc region residue 434 (See, e.g., U.S. Pat. No.7,371,826; Dall'Acqua, W. F., et al. J. Biol. Chem. 281 (2006)23514-23524).

Fc region residues critical to the mouse Fc-mouse FcRn interaction havebeen identified by site-directed mutagenesis (see e.g. Dall'Acqua, W.F., et al. J. Immunol 169 (2002) 5171-5180). Residues 1253, H310, H433,N434, and H435 (EU index numbering) are involved in the interaction(Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., etal., Int. Immunol. 13 (2001) 993; Kim, J. K., et al., Eur. J. Immunol.24 (1994) 542). Residues 1253, H310, and H435 were found to be criticalfor the interaction of human Fc with murine FcRn (Kim, J. K., et al.,Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRncomplex have shown that residues I253, S254, H435, and Y436 are crucialfor the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993;Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung,Y. A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants ofresidues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 havebeen reported and examined.

In certain aspects, an antibody variant comprises an Fc region with oneor more amino acid substitutions, which reduce FcRn binding, e.g.,substitutions at positions 253, and/or 310, and/or 435 of the Fc-region(EU numbering of residues). In certain aspects, the antibody variantcomprises an Fc region with the amino acid substitutions at positions253, 310 and 435. In one aspect, the substitutions are I253A, H310A andH435A in an Fc region derived from a human IgG1 Fc-region. See, e.g.,Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

In certain aspects, an antibody variant comprises an Fc region with oneor more amino acid substitutions, which reduce FcRn binding, e.g.,substitutions at positions 310, and/or 433, and/or 436 of the Fc region(EU numbering of residues). In certain aspects, the antibody variantcomprises an Fc region with the amino acid substitutions at positions310, 433 and 436. In one aspect, the substitutions are H310A, H433A andY436A in an Fc region derived from a human IgG1 Fc-region. (See, e.g.,WO 2014/177460 A1).

In certain aspects, an antibody variant comprises an Fc region with oneor more amino acid substitutions which increase FcRn binding, e.g.,substitutions at positions 252, and/or 254, and/or 256 of the Fc region(EU numbering of residues). In certain aspects, the antibody variantcomprises an Fc region with amino acid substitutions at positions 252,254, and 256. In one aspect, the substitutions are M252Y, S254T andT256E in an Fc region derived from a human IgG₁ Fc-region. See alsoDuncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260;5,624,821; and WO 94/29351 concerning other examples of Fc regionvariants.

The C-terminus of the heavy chain of the antibody as reported herein canbe a complete C-terminus ending with the amino acid residues PGK. TheC-terminus of the heavy chain can be a shortened C-terminus in which oneor two of the C terminal amino acid residues have been removed. In onepreferred aspect, the C-terminus of the heavy chain is a shortenedC-terminus ending PG. In one aspect of all aspects as reported herein,an antibody comprising a heavy chain including a C-terminal CH3 domainas specified herein, comprises the C-terminal glycine-lysine dipeptide(G446 and K447, EU index numbering of amino acid positions). In oneaspect of all aspects as reported herein, an antibody comprising a heavychain including a C-terminal CH3 domain, as specified herein, comprisesa C-terminal glycine residue (G446, EU index numbering of amino acidpositions).

c) Cysteine Engineered Antibody Variants

In certain aspects, it may be desirable to create cysteine engineeredantibodies, e.g., THIOMAB™ antibodies, in which one or more residues ofan antibody are substituted with cysteine residues. In particularaspects, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. Cysteine engineered antibodies may begenerated as described, e.g., in U.S. Pat. Nos. 7,521,541, 8,30,930,7,855,275, 9,000,130, or WO 2016040856.

7. Immunoconjugates

The invention also provides immunoconjugates comprising an antibody thatbinds to human VEGF and human IL-1beta as disclosed herein conjugated(chemically bonded) to one or more therapeutic agents such as cytotoxicagents, chemotherapeutic agents, drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one aspect, an immunoconjugate is an antibody-drug conjugate (ADC) inwhich an antibody is conjugated to one or more of the therapeutic agentsmentioned above. The antibody is typically connected to one or more ofthe therapeutic agents using linkers. An overview of ADC technologyincluding examples of therapeutic agents and drugs and linkers is setforth in Pharmacol Review 68:3-19 (2016).

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. For these methods one ormore isolated nucleic acid(s) encoding an antibody are provided.

In one aspect, isolated nucleic acids encoding an antibody of theinvention are provided.

In one aspect, a method of making an antibody that binds to human VEGFand human IL-1beta is provided, wherein the method comprises culturing ahost cell comprising nucleic acid(s) encoding the antibody, as providedabove, under conditions suitable for expression of the antibody, andoptionally recovering the antibody from the host cell (or host cellculture medium).

For recombinant production of an antibody that binds to human VEGF andhuman IL-1beta, nucleic acids encoding the antibody, e.g., as describedabove, are isolated and inserted into one or more vectors for furthercloning and/or expression in a host cell. Such nucleic acids may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of the antibody) or producedby recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press,Totowa, NJ (2003), pp. 245-254, describing expression of antibodyfragments in E. coli.) After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHOcells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004),pp. 255-268.

In one aspect, the host cell is eukaryotic, e.g., a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

C. Pharmaceutical Compositions

In a further aspect, provided are pharmaceutical compositions comprisingany of the antibodies provided herein, e.g., for use in any of the belowtherapeutic methods. In one aspect, a pharmaceutical compositioncomprises any of the antibodies provided herein and a pharmaceuticallyacceptable carrier. In another aspect, a pharmaceutical compositioncomprises any of the antibodies provided herein and at least oneadditional therapeutic agent, e.g., as described below.

Pharmaceutical compositions of an antibody that binds to human VEGF andhuman IL-1beta as described herein are prepared by mixing such antibodyhaving the desired degree of purity with one or more optionalpharmaceutically acceptable carriers (Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedcompositions or aqueous solutions. Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such ashistidine, phosphate, citrate, acetate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, aredescribed in US Patent Publication Nos. 2005/0260186 and 2006/0104968.In one aspect, a sHASEGP is combined with one or more additionalglycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody compositions are described in U.S. Pat.No. 6,267,958. Aqueous antibody compositions include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter compositionsincluding a histidine-acetate buffer.

The pharmaceutical composition herein may also contain more than oneactive ingredients as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Pharmaceutical compositions for sustained-release may be prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules.

The pharmaceutical compositions to be used for in vivo administrationare generally sterile. Sterility may be readily accomplished, e.g., byfiltration through sterile filtration membranes.

D. Therapeutic Methods and Routes of Administration

Any of the antibodies that bind to human VEGF and human IL-1betaprovided herein may be used in therapeutic methods.

In one aspect, an antibody that binds to human VEGF and human IL-1betafor use as a medicament is provided. In further aspects, an antibodythat binds to human VEGF and human IL-1beta for use in treating avascular disease is provided. In certain aspects, an antibody that bindsto human VEGF and human IL-1beta for use in a method of treatment isprovided. In certain aspects, the invention provides an antibody thatbinds to human VEGF and human IL-1beta for use in a method of treatingan individual having a vascular disease comprising administering to theindividual an effective amount of the antibody that binds to human VEGFand human IL-1beta. In one such aspect, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent (e.g., one, two, three, four, five, or sixadditional therapeutic agents), e.g., as described below. In furtheraspects, the invention provides an antibody that binds to human VEGF andhuman IL-1beta for use in inhibiting angiogenesis. In certain aspects,the invention provides an antibody that binds to human VEGF and humanIL-1beta for use in a method inhibiting angiogenesis in an individualcomprising administering to the individual an effective amount of theantibody that binds to human VEGF and human IL-1beta to inhibitangiogenesis. An “individual” according to any of the above aspects ispreferably a human.

In further aspects, an antibody that binds to human VEGF and humanIL-1beta for use in treating an ocular disease is provided. In oneembodiment the ocular disease is selected from AMD (in one embodimentwet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy(GA)), macular degeneration, macular edema, DME (in one embodimentfocal, non-center DME and diffuse, center-involved DME), retinopathy,diabetic retinopathy (DR) (in one embodiment proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (inone embodiment central (CRVO) and branched (BRVO) forms), CNV (in oneembodiment myopic CNV), corneal neovascularization, diseases associatedwith corneal neovascularization, retinal neovascularization, diseasesassociated with retinal/choroidal neovascularization, central serousretinopathy (CSR), pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, FEVR, Coats' disease, Norrie Disease, retinalabnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG),subconjunctival hemorrhage, rubeosis, ocular neovascular disease,neovascular glaucoma, retinitis pigmentosa (RP), hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, cystoid macular edema (CME), vasculitis, papilloedema,retinitis, including but not limited to CMV retinitis, ocular melanoma,retinal blastoma, conjunctivitis (in one embodiment infectiousconjunctivitis and non-infectious (in one embodiment allergic)conjunctivitis), Leber congenital amaurosis (also known as Leber'scongenital amaurosis or LCA), uveitis (including infectious andnon-infectious uveitis), choroiditis (in one embodiment multifocalchoroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eyeinjury, Sjögren's disease, and other ophthalmic diseases wherein thedisease or disease is associated with ocular neovascularization,vascular leakage, and/or retinal edema or retinal atrophy. In oneembodiment the ocular disease is selected from AMD (in one embodimentwet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy(GA)), macular degeneration, macular edema, DME (in one embodimentfocal, non-center DME and diffuse, center-involved DME), retinopathy,diabetic retinopathy (DR) (in one embodiment proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR.

In a further aspect, the invention provides for the use of an antibodythat binds to human VEGF and human IL-1beta in the manufacture orpreparation of a medicament. In one aspect, the medicament is fortreatment of a vascular disease. In a further aspect, the medicament isfor use in a method of treating a vascular disease comprisingadministering to an individual having a vascular disease an effectiveamount of the medicament. In one such aspect, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below.

In one aspect, the medicament is for treatment of an ocular disease. Ina further aspect, the medicament is for use in a method of treating anocular disease comprising administering to an individual having anocular disease an effective amount of the medicament. In one suchaspect, the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, e.g., asdescribed below.

In a further aspect, the invention provides a method for treating avascular disease. In one aspect, the method comprises administering toan individual having such vascular disease an effective amount of anantibody that binds to human VEGF and human IL-1 beta. In one suchaspect, the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, asdescribed below.

In a further aspect, the invention provides a method for treating anocular disease. In one aspect, the method comprises administering to anindividual having such ocular disease an effective amount of an antibodythat binds to human VEGF and human IL-1beta. In one such aspect, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, as described below.

An “individual” according to any of the above aspects may be a human.

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the antibodies that bind to human VEGF and human IL-1beta provided herein, e.g., for use in any of the above therapeuticmethods. In one aspect, a pharmaceutical composition comprises any ofthe antibodies that bind to human VEGF and human IL-1 beta providedherein and a pharmaceutically acceptable carrier. In another aspect, apharmaceutical composition comprises any of the antibodies that bind tohuman VEGF and human IL-1beta provided herein and at least oneadditional therapeutic agent, e.g., as described below.

Antibodies of the invention can be administered alone or used in acombination therapy. For instance, the combination therapy includesadministering an antibody of the invention and administering at leastone additional therapeutic agent (e.g. one, two, three, four, five, orsix additional therapeutic agents).

For example, in certain embodiments, any of the preceding methodsfurther comprises administering one or more additional compounds. Incertain embodiments, the antibody that binds to human VEGF and humanIL-1beta provided herein is administered simultaneously with theadditional compound(s). In certain embodiments, the antibody that bindsto human VEGF and human IL-1beta is administered before or after theadditional compound(s). In certain embodiments, the additional compoundbinds to a second biological molecule selected from the group consistingof IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2; S1P;integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and proteins genetically linked to AMD risk, such ascomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1;TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In certain embodiments,the additional compound is an antibody or antigen-binding fragmentthereof.

In certain embodiments according to (or as applied to) any of theembodiments above, the ocular disoder is an intraocular neovasculardisease selected from the group consisting of proliferativeretinopathies, choroidal neovascularization (CNV), age-related maculardegeneration (AMD), diabetic and other ischemia-related retinopathies,diabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (RVO), including CRVOand BRVO, comeal neovascularization, retinal neovascularization, andretinopathy of prematurity (ROP).

In some instances, an antibody that binds to human VEGF and humanIL-1beta provided herein may be administered in combination with atleast one additional therapeutic agent for treatment of an oculardisorder, for example, an ocular disorder described herein (e.g., AMD(e.g., wet AMD), DME, DR, RVO, or GA). Exemplary additional therapeuticagents for combination therapy for treatment of ocular disordersinclude, without limitation, anti-angiogenic agents, such as VEGFantagonists, including, for example, anti-VEGF antibodies (e.g., theanti-VEGF Fab LUCENTIS® (ranibizumab)), soluble receptor fusion proteins(e.g., the recombinant soluble receptor fusion protein EYLEA®(aflibercept, also known as VEGF Trap Eye; Regeneron/Aventis)), aptamers(e.g., the anti-VEGF pegylated aptamer MACUGEN® (pegaptanib sodium;NeXstar Pharmaceuticals/OSI Pharmaceuticals)), and VEGFR tyrosine kinaseinhibitors (e.g.,4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT®(sunitinib)); Tryptophanyl-tRNA synthetase (TrpRS); squalamine; RETAANE®(anecortave acetate for depot suspension; Alcon, Inc.); CombretastatinA4 Prodrug (CA4P); MIFEPREX® (mifepristone-ru486); subtenontriamcinolone acetonide; intravitreal crystalline triamcinoloneacetonide; matrix metalloproteinase inhibitors (e.g., Prinomastat(AG3340; Pfizer)); fluocinolone acetonide (including fluocinoloneintraocular implant; Bausch & Lomb/Control Delivery Systems); linomide;inhibitors of integrin β3 function; angiostatin, and combinationsthereof. These and other therapeutic agents that can be administered incombination with an antibody that binds to human VEGF and human IL-1betaof the invention are described, for example, in U.S. Patent ApplicationNo. US 2014/0017244, which is incorporated herein by reference in itsentirety.

Further examples of additional therapeutic agents that can be used incombination with an antibody that binds to human VEGF and humanIL-1betaas provided herein for treatment of an ocular disorder (e.g.,AMD, DME, DR, RVO, or GA), include, but are not limited to, VISUDYNE®(verteporfin; a light-activated drug that is typically used inconjunction with photodynamic therapy with a non-thermal laser), PKC412,Endovion (NS 3728; NeuroSearch A/S), neurotrophic factors (e.g., glialderived neurotrophic factor (GDNF) and ciliary neurotrophic factor(CNTF)), diltiazem, dorzolamide, PHOTOTROP®, 9-cis-retinal, eyemedication (e.g., phospholine iodide, echothiophate, or carbonicanhydrase inhibitors), veovastat (AE-941; AEterna Laboratories, Inc.),Sirna-027 (AGF-745; Sima Therapeutics, Inc.), neurotrophins (including,by way of example only, NT-4/5, Genentech), CandS (AcuityPharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrinantagonists (including those from Jerini AG and Abbott Laboratories),EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (asused, for example, by EntreMed, Inc.), cardiotrophin-1 (Genentech),2-methoxyestradiol (Allergan/Oculex), DL-8234 (Toray Industries),NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan),LYN-002 (Lynkeus Biotech), microalgal compound (Aquasearch/Albany, MeraPharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (HamamatsuPhotonics), TGF-beta 2 (Genzyme/Celtrix), tyrosine kinase inhibitors(e.g., those from Allergan, SUGEN, or Pfizer), NX-278-L (NeXstarPharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal cellganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazolederivatives (Texas A&M University System), KP-102 (KrenitskyPharmaceuticals), cyclosporin A, therapeutic agents used in photodynamictherapy (e.g., VISUDYNE®; receptor-targeted PDT, Bristol-Myers Squibb,Co.; porfimer sodium for injection with PDT; verteporfin, QLT Inc.;rostaporfin with PDT, Miravent Medical Technologies; talaporfin sodiumwith PDT, Nippon Petroleum; and motexafin lutetium, Pharmacyclics,Inc.), antisense oligonucleotides (including, by way of example,products tested by Novagali Pharma SA and ISIS-13650, lonisPharmaceuticals), and combinations thereof.

An antibody that binds to human VEGF and human IL-1beta as providedherein may be administered in combination with a therapy or surgicalprocedure for treatment of an ocular disorder (e.g., AMD, DME, DR, RVO,or GA), including, for example, laser photocoagulation (e.g., panretinalphotocoagulation (PRP)), drusen lasering, macular hole surgery, maculartranslocation surgery, implantable miniature telescopes, PHI-motionangiography (also known as micro-laser therapy and feeder vesseltreatment), proton beam therapy, microstimulation therapy, retinaldetachment and vitreous surgery, scleral buckle, submacular surgery,transpupillary thermotherapy, photosystem I therapy, use of RNAinterference (RNAi), extracorporeal rheopheresis (also known as membranedifferential filtration and rheotherapy), microchip implantation, stemcell therapy, gene replacement therapy, ribozyme gene therapy (includinggene therapy for hypoxia response element, Oxford Biomedica; Lentipak,Genetix; and PDEF gene therapy, GenVec), photoreceptor/retinal cellstransplantation (including transplantable retinal epithelial cells,Diacrin, Inc.; retinal cell transplant, e.g., Astellas Pharma US, Inc.,ReNeuron, CHA Biotech), acupuncture, and combinations thereof.

In some instances, an antibody that binds to human VEGF and humanIL-1beta can be administered in combination with an anti-angiogenicagent for treatment of an ocular disorder (e.g., AMD, DME, DR, RVO, orGA). Any suitable anti-angiogenic agent can be used in combination withan antibody that binds to human VEGF and human IL-1 beta of theinvention, including, but not limited to, those listed by Carmeliet etal. Nature 407:249-257, 2000. In some embodiments, the anti-angiogenicagent is a VEGF antagonist, including, but not limited to, an anti-VEGFantibody (e.g., the anti-VEGF Fab LUCENTIS® (ranibizumab), RTH-258(formerly ESBA-1008, an anti-VEGF single-chain antibody fragment;Novartis), or a bispecific anti-VEGF antibody (e.g., ananti-VEGF/anti-angiopoeitin 2 bispecific antibody such as faricimab;Roche)), a soluble recombinant receptor fusion protein (e.g., EYLEA®(aflibercept)), a VEGF variant, a soluble VEGFR fragment, an aptamercapable of blocking VEGF (e.g., pegaptanib) or VEGFR, a neutralizinganti-VEGFR antibody, a small molecule inhibitor of VEGFR tyrosinekinases, an anti-VEGF DARPin® (e.g., abicipar pegol, Molecular PartnersAG/Allergan), a small interfering RNAs which inhibits expression of VEGFor VEGFR, a VEGFR tyrosine kinase inhibitor (e.g.,4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT®(sunitinib)), and combinations thereof.

Other suitable anti-angiogenic agents that may be administered incombination with an antibody that binds to human VEGF and human IL-1betaas provided herein for treatment of an ocular disorder (e.g., AMD, DME,DR, RVO, or GA) include corticosteroids, angiostatic steroids,anecortave acetate, angiostatin, endostatin, tyrosine kinase inhibitors,matrix metalloproteinase (MMP) inhibitors, insulin-like growthfactor-binding protein 3 (IGFBP3), stromal derived factor (SDF-1)antagonists (e.g., anti-SDF-1 antibodies), pigment epithelium-derivedfactor (PEDF), gamma-secretase, Delta-like ligand 4, integrinantagonists, hypoxia-inducible factor (HIF)-1α antagonists, proteinkinase CK2 antagonists, agents that inhibit stem cell (e.g., endothelialprogenitor cell) homing to the site of neovascularization (e.g., ananti-vascular endothelial cadherin (CD-144) antibody and/or ananti-SDF-1 antibody), and combinations thereof.

In a further example, in some instances, an antibody that binds to humanVEGF and human IL-1beta, and/or polymeric formulation thereof, can beadministered in combination with an agent that has activity againstneovascularization for treatment of an ocular disorder (e.g., AMD, DME,DR, RVO, or GA), such as an anti-inflammatory drug, a mammalian targetof rapamycin (mTOR) inhibitor (e.g., rapamycin, AFINITOR® (everolimus),and TORISEL® (temsirolimus)), cyclosporine, a tumor necrosis factor(TNF) antagonist (e.g., an anti-TNFα antibody or antigen-bindingfragment thereof (e.g., infliximab, adalimumab, certolizumab pegol, andgolimumab) or a soluble receptor fusion protein (e.g., etanercept)), ananti-complement agent, a nonsteroidal antiinflammatory agent (NSAID), orcombinations thereof.

In a still further example, in some instances, an antibody that binds tohuman VEGF and human IL-1beta can be administered in combination with anagent that is neuroprotective and can potentially reduce the progressionof dry AMD to wet AMD, such as the class of drugs called the“neurosteroids,” which include drugs such as dehydroepiandrosterone(DHEA) (brand names: PRASTERA™ and FIDELIN®), dehydroepiandrosteronesulfate, and pregnenolone sulfate.

Any suitable AMD therapeutic agent can be administered as an additionaltherapeutic agent in combination with an antibody that binds to humanVEGF and human IL-1beta as provided herein for treatment of an oculardisorder (e.g., AMD, DME, DR, RVO, or GA), including, but not limitedto, a VEGF antagonist, for example, an anti-VEGF antibody (e.g.,LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGFsingle-chain antibody fragment; Novartis), or a bispecific anti-VEGFantibody (e.g., an anti-VEGF/anti-angiopoeitin 2 bispecific antibodysuch as faricimab; Roche)), a soluble VEGF receptor fusion protein(e.g., EYLEA® (aflibercept)), an anti-VEGF DARPin® (e.g., abiciparpegol; Molecular Partners AG/Allergan), or an anti-VEGF aptamer (e.g.,MACUGEN® (pegaptanib sodium)); a platelet-derived growth factor (PDGF)antagonist, for example, an anti-PDGF antibody, an anti-PDGFR antibody(e.g., REGN2176-3), an anti-PDGF-BB pegylated aptamer (e.g., FOVISTA®;Ophthotech/Novartis), a soluble PDGFR receptor fusion protein, or a dualPDGF/VEGF antagonist (e.g., a small molecule inhibitor (e.g., DE-120(Santen) or X-82 (TyrogeneX)) or a bispecific anti-PDGF/anti-VEGFantibody)); VISUDYNE® (verteporfin) in combination with photodynamictherapy; an antioxidant; a complement system antagonist, for example, acomplement factor C5 antagonist (e.g., a small molecule inhitor (e.g.,ARC-1905; Opthotech) or an anti-C5 antibody (e.g., LFG-316; Novartis), aproperdin antagonist (e.g., an anti-properdin antibody, e.g., CLG-561;Alcon), or a complement factor D antagonist (e.g., an anti-complementfactor D antibody, e.g., lampalizumab; Roche)); a C3 blocking peptide(e.g., APL-2, Appellis); a visual cycle modifier (e.g., emixustathydrochloride); squalamine (e.g., OHR-102; Ohr Pharmaceutical); vitaminand mineral supplements (e.g., those described in the Age-Related EyeDisease Study 1 (AREDS1; zinc and/or antioxidants) and Study 2 (AREDS2;zinc, antioxidants, lutein, zeaxanthin, and/or omega-3 fatty acids)); acell-based therapy, for example, NT-501 (Renexus); PH-05206388 (Pfizer),huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cordstem cell line; Janssen), OpRegen (suspension of RPE cells; Cell CureNeurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics);a tissue factor antagonist (e.g., hI-conl; Iconic Therapeutics); analpha-adrenergic receptor agonist (e.g., brimonidine tartrate;Allergan); a peptide vaccine (e.g., S-646240; Shionogi); an amyloid betaantagonist (e.g., an anti-beta amyloid monoclonal antibody, e.g.,GSK-933776); an SIP antagonist (e.g., an anti-S1P antibody, e.g.,iSONEP™; Lpath Inc); a ROBO4 antagonist (e.g., an anti-ROBO4 antibody,e.g., DS-7080a; Daiichi Sankyo); a lentiviral vector expressingendostatin and angiostatin (e.g., RetinoStat); and any combinationthereof. In some instances, AMD therapeutic agents (including any of thepreceding AMD therapeutic agents) can be co-formulated. For example, theanti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept(EYLEA®). In some instances, such a co-formulation can be administeredin combination with an antibody that binds to human VEGF and humanIL-1beta of the invention. In some instances, the ocular disorder is AMD(e.g., wet AMD).

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with LUCENTIS® (ranibizumab) fortreatment of an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). Insome instances, the ocular disorder is AMD (e.g., wet AMD). In someinstances, the ocular disorder is GA.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with EYLEA® (aflibercept) fortreatment of an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). Insome instances, the ocular disorder is AMD (e.g., wet AMD). In someinstances, the ocular disorder is GA.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with MACUGEN® (pegaptanib sodium) fortreatment of an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). Insome instances, the ocular disorder is AMD (e.g., wet AMD). In someinstances, the ocular disorder is GA.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with VISUDYNE® (verteporfin) incombination with photodynamic therapy for treatment of an oculardisorder (e.g., AMD, DME, DR, RVO, or GA). In some instances, the oculardisorder is AMD (e.g., wet AMD). In some instances, the ocular disorderis GA.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with a PDGF antagonist for treatmentof an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). Exemplary PDGFantagonists which may be used in combination with an antibody that bindsto human VEGF and human IL-1beta of the invention include an anti-PDGFantibody, an anti-PDGFR antibody, a small molecule inhibitor (e.g.,squalamine), an anti-PDGF-B pegylated aptamer such as FOVISTA® (E10030;Ophthotech/Novartis), or a dual PDGF/VEGF antagonist (e.g., a smallmolecule inhibitor (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or abispecific anti-PDGF/anti-VEGF antibody). For example, FOVISTA® can beadministered as an adjunct therapy to an antibody that binds to humanVEGF and human IL-1beta of the invention. OHR-102 can be administered incombination with VEGF antagonists such as LUCENTIS® or EYLEA®. In someembodiments, an antibody that binds to human VEGF and human IL-1beta ofthe invention can be administered in combination with OHR-102,LUCENTIS®, and/or EYLEA®. In some instances, the ocular disorder is AMD(e.g., wet AMD). In some instances, the ocular disorder is GA.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with RTH-258 for treatment of anocular disorder (e.g., AMD, DME, DR, RVO, or GA). RTH-258 can beadministered, for example, by intravitreal injection or eye infusion. Insome instances, the ocular disorder is AMD (e.g., wet AMD). In someinstances, the ocular disorder is GA.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with abicipar pegol for treatment ofan ocular disorder (e.g., AMD, DME, DR, RVO, or GA). In some instances,the ocular disorder is AMD (e.g., wet AMD). In some instances, theocular disorder is GA.

Any suitable DME and/or DR therapeutic agent can be administered incombination with an antibody that binds to human VEGF and human IL-1betaof the invention for treatment of an ocular disorder (e.g., AMD, DME,DR, RVO, or GA), including, but not limited, to a VEGF antagonist (e.g.,LUCENTIS® or EYLEA®), a corticosteroid (e.g., a corticosteroid implant(e.g., OZURDEX® (dexamethasone intravitreal implant) or ILUVIEN®(fluocinolone acetonide intravitreal implant)) or a corticosteroidformulated for administration by intravitreal injection (e.g.,triamcinolone acetonide)), or combinations thereof. In some instances,the ocular disorder is DME and/or DR.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with LUCENTIS® (ranibizumab) fortreatment of DME and/or DR (e.g., NPDR or PDR).

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with EYLEA® (aflibercept) fortreatment of DME and/or DR (e.g., NPDR or PDR).

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with OZURDEX® (dexamethasoneintravitreal implant) for treatment of DME and/or DR.

An antibody that binds to human VEGF and human IL-1beta of the inventioncan be administered in combination with ILUVIEN® (dexamethasoneintravitreal implant) for treatment of DME and/or DR.

In some cases, the TAO/PRN treatment regimen or TAE treatment regimenmay be used to administer an AMD therapeutic agent (e.g., ranibizumab oraflibercept) in combination with an antibody that binds to human VEGFand human IL-1beta of the invention, and/or polymeric formulationthereof. In some instances, the ocular disorder is AMD (e.g., wet AMD).In some instances, the ocular disorder is GA.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody that binds to human VEGF and humanIL-1beta of the invention can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent or agents.In one embodiment, administration of the antibody that binds to humanVEGF and human IL-1beta of the invention and administration of anadditional therapeutic agent occur within about one, two, three, four,or five months, or within about one, two or three weeks, or within aboutone, two, three, four, five, or six days, of each other.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.,by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the pharmaceutical composition, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.,every week or every three weeks (e.g., such that the patient receivesfrom about two to about twenty, or, e.g., about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. The progress of this therapy is easilymonitored by conventional techniques and assays.

E. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this aspect of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

3. Specific Embodiments of the Invention

In the following specific embodiments of the invention are listed.

-   -   1. An antibody that binds to human VEGF and to human IL-1beta,        comprising a VEGF paratope and an IL-1beta paratope within one        cognate pair of a variable light chain domain (VL domain) and a        variable heavy chain domain (VH domain), wherein the VEGF        paratope comprises amino acid residues from CDR-H2, CDR-L1 and        CDR-L3 of the antibody, wherein the IL-1beta paratope comprises        amino acid residues from the CDR-H1, CDR-H3 and CDR-L2 of the        antibody.    -   2. An antibody that binds to human VEGF and to human IL-1beta,        comprising a VEGF paratope and an IL-1beta paratope within one        cognate pair of a variable light chain domain (VL domain) and a        variable heavy chain domain (VH domain), wherein the pair of the        variable light chain domain and the variable heavy chain domain        simultaneously binds to human VEGF and human IL-1beta.    -   3. An antibody that binds to human VEGF and to human IL-1beta,        comprising a VEGF paratope and an IL-1beta paratope within one        cognate pair of a variable light chain domain (VL domain) and a        variable heavy chain domain (VH domain), wherein none of the        amino acids that are comprised in the VEGF paratope are        comprised in the IL-1beta paratope.    -   4. An antibody that binds to human VEGF and to human IL-1beta,        comprising a VEGF paratope and an IL-1beta paratope within one        cognate pair of a variable light chain domain (VL domain) and a        variable heavy chain domain (VH domain), wherein the antibody        binds to the same epitope on human VEGF and to the same epitope        on human IL-1beta as an antibody with a variable heavy chain        domain of SEQ ID NO: 11 and a variable light chain domain of SEQ        ID NO: 12.    -   5. An antibody that binds to human VEGF and to human IL-1beta,        comprising a VEGF paratope and an IL-1beta paratope within one        cognate pair of a variable light chain domain (VL domain) and a        variable heavy chain domain (VH domain), wherein        -   the VEGF paratope comprises amino acid residues from CDR-H2,            CDR-L1 and CDR-L3 of the antibody, wherein the IL-1beta            paratope comprises amino acid residues from the CDR-H1,            CDR-H3 and CDR-L2 of the antibody; and/or        -   the pair of the variable light chain domain and the variable            heavy chain domain simultaneously binds to human VEGF and            human IL-1beta; and/or        -   none of the amino acids that are comprised in the VEGF            paratope are comprised in the IL-1beta paratope; and/or        -   the antibody binds to the same epitope on human VEGF and to            the same epitope on human IL-1beta as an antibody with a            variable heavy chain domain of SEQ ID NO: 11 and a variable            light chain domain of SEQ ID NO: 12; and/or        -   an antibody Fab fragment of the antibody binds (i) to human            VEGF121 with a K_(D) of less than 10 pM as measured by            surface plasmon resonance, and (ii) to human IL-1beta with a            K_(D) of less than 30 pM as measured by surface plasmon            resonance; and/or        -   an antibody Fab fragment of the antibody exhibits an            aggregation onset temperature of more than 70° C.; and/or        -   an antibody Fab fragment of the antibody exhibits a melting            temperature of more than 80° C. as measured by dynamic light            scattering; and/or        -   binding of an antibody Fab fragment of the antibody to human            VEGF inhibits binding of VEGF to VEGFR2 with an IC50 of less            than 50 nM as measured by surface plasmon resonance; and            wherein binding of an antibody Fab fragment of the antibody            to human IL-1beta inhibits binding of IL-1beta to IL-1betaR1            with an IC50 of less than 30 nM as measured by surface            plasmon resonance.    -   6. The antibody of one of one of the preceding embodiments,        wherein the antibody comprises a VH domain comprising (a) CDR-H1        comprising the amino acid sequence of SEQ ID NO:13, (b) CDR-H2        comprising the amino acid sequence of SEQ ID NO:14, and (c)        CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and a        VL domain comprising (d) CDR-L1 comprising the amino acid        sequence of SEQ ID NO:16, (e) CDR-L2 comprising the amino acid        sequence of SEQ ID NO:17, and (f) CDR-L3 comprising the amino        acid sequence of SEQ ID NO:8.    -   7. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8.    -   8. The antibody of one of one of the preceding embodiments,        wherein the antibody comprises a VH domain comprising (a) CDR-H1        comprising the amino acid sequence of SEQ ID NO:13, (b) CDR-H2        comprising the amino acid sequence of SEQ ID NO:14, (c) CDR-H3        comprising the amino acid sequence of SEQ ID NO:15, (d) a human        heavy chain framework with (i) FR1 comprising amino acid        residues E2, G26, V28, and K30, (ii) FR3 comprising amino acid        residues R66, R83, and K94; and a VL domain comprising (e)        CDR-L1 comprising the amino acid sequence of SEQ ID NO:16, (f)        CDR-L2 comprising the amino acid sequence of SEQ ID NO:17, (g)        CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,        and (h) a human light chain framework with (i) FR1 comprising        amino acid residue I2, (ii) FR2 comprising amino acid residue        Y49, (iii) FR3 comprising amino acid residues G57, E67, D68, and        Q69, wherein the numbering of the VH and VL domains is according        to the Kabat numbering system.    -   9. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, (c) CDR-H3 comprising the amino acid sequence of SEQ        ID NO:15, (d) a human heavy chain framework with (i) FR1        comprising amino acid residues E2, G26, V28, and K30, (ii) FR3        comprising amino acid residues R66, R83, and K94; and a VL        domain comprising (e) CDR-L1 comprising the amino acid sequence        of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequence        of SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence        of SEQ ID NO: 8, and (h) a human light chain framework with (i)        FR1 comprising amino acid residue I2, (ii) FR2 comprising amino        acid residue Y49, (iii) FR3 comprising amino acid residues G57,        E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system.    -   10. The antibody of one of the preceding embodiments, comprising        a VH domain comprising amino acid residues E2, G26, V28, K30,        W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66,        R83, K94, D95, V96, F98, and D101, and a VL domain comprising        amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50,        Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94, and Y96,        wherein the numbering of the VH and VL domains is according to        the Kabat numbering system.    -   11. The antibody of embodiment 10, comprising        -   a VEGF paratope comprising the following amino acid residues            in the VH domain D55, H56, Y58, T61, K62, F63, I64, R66, and            R83, and the following amino acid residues in the VL domain            12, Y27, W27a, S27c, S27d, E67, D68, Q69, R92, Y93, H94, and            Y96; and        -   an IL-1beta paratope comprising the following amino acid            residues in the VH domain E2, G26, V28, K30, W31, N35b,            D35c, K52a, K94, D95, V96, F98, and D101, and the following            amino acid residues in the VL domain L32, Y49, D50, Y53,            K54, L56, G57, Y91.    -   12. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising within one pair of a VL domain and a        VH domain: (i) a VH domain comprising amino acid residues E2,        G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62,        F63, I64, R66, R83, K94, D95, V96, F98, and D101, and (ii) a VL        domain comprising amino acid residues I2, Y27, W27a, S27c, S27d,        L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93,        H94, and Y96, wherein the numbering of the VH and VL domains is        according to the Kabat numbering system.    -   13. The antibody of embodiment 12, comprising        -   a VEGF paratope comprising the following amino acid residues            in the VH domain: D55, H56, Y58, T61, K62, F63, I64, R66,            and R83, and the following amino acid residues in the VL            domain: 12, Y27, W27a, S27c, S27d, E67, D68, Q69, R92, Y93,            H94, and Y96; and        -   an IL-1beta paratope comprising the following amino acid            residues in the VH domain: E2, G26, V28, K30, W31, N35b,            D35c, K52a, K94, D95, V96, F98, and D101, and the following            amino acid residues in the VL domain: L32, Y49, D50, Y53,            K54, L56, G57, Y91.    -   14. The antibody of any one of the preceding embodiments,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11; and (b) a VL domain comprising an amino acid        sequence having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:12.    -   15. The antibody of any one of the preceding embodiments,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11, wherein the VH domain comprises amino acid        residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56,        Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98, and D101;        and (b) a VL domain comprising an amino acid sequence having at        least 90% sequence identity to the amino acid sequence of SEQ ID        NO:12, wherein the VL domain comprises amino acid residues I2,        Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67,        D68, Q69, Y91, R92, Y93, H94, and Y96, wherein the numbering of        the VH and VL domains is according to the Kabat numbering        system.    -   16. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11; and (b) a VL domain comprising an amino acid        sequence having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:12.    -   17. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11, wherein the VH domain comprises amino acid        residues E2, G26, V28, K30, R66, R83, and K94; and (b) a VL        domain comprising an amino acid sequence having at least 90%        sequence identity to the amino acid sequence of SEQ ID NO:12,        wherein the VL domain comprises amino acid residues I2, Y49,        G57, E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system.    -   18. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, (c) CDR-H3 comprising the amino acid sequence of SEQ        ID NO:15, (d) a human heavy chain framework with (i) FR1        comprising amino acid residues E2, G26, V28, and K30, (ii) FR3        comprising amino acid residues R66, R83, and K94: and a VL        domain comprising (e) CDR-L1 comprising the amino acid sequence        of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequence        of SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence        of SEQ ID NO: 8, and (h) a human light chain framework with (i)        FR1 comprising amino acid residue I2, (ii) FR2 comprising amino        acid residue Y49, (iii) FR3 comprising amino acid residues G57,        E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11; and (b) a VL domain comprising an amino acid        sequence having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:12.    -   19. The antibody of any one of the preceding embodiments,        comprising (a) a VH domain comprising an amino acid sequence of        SEQ ID NO:11 with up to 15 amino acid substitutions; and (b) a        variable light chain domain comprising an amino acid sequence of        SEQ ID NO:12 with up to 15 amino acid substitutions.    -   20. The antibody of any one of the preceding embodiments,        comprising (a) a VH domain comprising an amino acid sequence of        SEQ ID NO:11 with up to 15 amino acid substitutions, wherein the        amino acid substitutions are located at positions 3 to 25, 36 to        49, 97 to 82c, 84 to 93, or 103 to 113 of SEQ ID NO:11; and (b)        a variable light chain domain comprising an amino acid sequence        of SEQ ID NO:12 with up to 15 amino acid substitutions, wherein        the amino acid substitutions are located at positions 1, 4, 6, 8        to 23, 35 to 48, 58 to 66, 70 to 88, or 98 to 107 of SEQ ID        NO:12, wherein the numbering of the VH and VL domains is        according to the Kabat numbering system.    -   21. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8,        comprising (a) a VH domain comprising an amino acid sequence of        SEQ ID NO:11 with up to 15 amino acid substitutions; and (b) a        variable light chain domain comprising an amino acid sequence of        SEQ ID NO:12 with up to 15 amino acid substitutions.    -   22. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, (c) CDR-H3 comprising the amino acid sequence of SEQ        ID NO:15, (d) a human heavy chain framework with (i) FR1        comprising amino acid residues E2, G26, V28, and K30, (ii) FR3        comprising amino acid residues R66, R83, and K94. and a VL        domain comprising (e) CDR-L1 comprising the amino acid sequence        of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequence        of SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence        of SEQ ID NO: 8, and (h) a human light chain framework with (i)        FR1 comprising amino acid residue I2, (ii) FR2 comprising amino        acid residue Y49, (iii) FR3 comprising amino acid residues G57,        E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system, and        comprising (a) a VH domain comprising an amino acid sequence of        SEQ ID NO:11 with up to 15 amino acid substitutions; and (b) a        variable light chain domain comprising an amino acid sequence of        SEQ ID NO:12 with up to 15 amino acid substitutions.    -   23. The antibody of any one of the preceding embodiments,        comprising a VH sequence of SEQ ID NO:11 and a VL sequence of        SEQ ID NO:12.    -   24. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising a VH sequence of SEQ ID NO:11 and a        VL sequence of SEQ ID NO:12.    -   25. The antibody of any one of the preceding embodiments,        comprising a heavy chain amino acid sequence of SEQ ID NO:20 and        a light chain amino acid sequence of SEQ ID NO:19.    -   26. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising a heavy chain amino acid sequence of        SEQ ID NO:20 and a light chain amino acid sequence of SEQ ID        NO:19.    -   27. The antibody of any one of the preceding embodiments,        comprising a heavy chain amino acid sequence of SEQ ID NO:18 and        a light chain amino acid sequence of SEQ ID NO:19.    -   28. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising a heavy chain amino acid sequence of        SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID        NO:19.    -   29. The antibody of any one of the preceding embodiments,        wherein an antibody Fab fragment of the antibody binds (i) to        human VEGF121 with a K_(D) of less than 10 pM as measured by        surface plasmon resonance, and (ii) to human IL-1beta with a        K_(D) of less than 30 pM as measured by surface plasmon        resonance.    -   30. An antibody that specifically binds to human VEGF and to        human IL-1beta wherein an antibody Fab fragment of the antibody        binds (i) to human VEGF121 with a K_(D) of less than 10 pM as        measured by surface plasmon resonance, and (ii) to human        IL-1beta with a K_(D) of less than 30 pM as measured by surface        plasmon resonance.    -   31. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8, wherein an        antibody Fab fragment of the antibody binds (i) to human VEGF121        with a K_(D) of less than 10 pM as measured by surface plasmon        resonance, and (ii) to human IL-1beta with a K_(D) of less than        30 pM as measured by surface plasmon resonance.    -   32. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, (c) CDR-H3 comprising the amino acid sequence of SEQ        ID NO:15, (d) a human heavy chain framework with (i) FR1        comprising amino acid residues E2, G26, V28, and K30, (ii) FR3        comprising amino acid residues R66, R83, and K94: and a VL        domain comprising (e) CDR-L1 comprising the amino acid sequence        of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequence        of SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence        of SEQ ID NO: 8, and (h) a human light chain framework with (i)        FR1 comprising amino acid residue I2, (ii) FR2 comprising amino        acid residue Y49, (iii) FR3 comprising amino acid residues G57,        E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system, wherein an        antibody Fab fragment of the antibody binds (i) to human VEGF121        with a K_(D) of less than 10 pM as measured by surface plasmon        resonance, and (ii) to human IL-1beta with a K_(D) of less than        30 pM as measured by surface plasmon resonance.    -   33. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising within one pair of a VH and VL        domain: (i) a VH domain comprising amino acid residues E2, G26,        V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63,        I64, R66, R83, K94, D95, V96, F98, and D101, and (ii) VL domain        comprising amino acid residues I2, Y27, W27a, S27c, S27d, L32,        Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94,        and Y96, wherein the numbering of the VH and VL domains is        according to the Kabat numbering system, wherein an antibody Fab        fragment of the antibody binds (i) to human VEGF121 with a K_(D)        of less than 10 pM as measured by surface plasmon resonance,        and (ii) to human IL-1beta with a K_(D) of less than 30 pM as        measured by surface plasmon resonance.    -   34. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11; and (b) a VL domain comprising an amino acid        sequence having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:12; wherein an antibody Fab fragment of        the antibody binds (i) to human VEGF121 with a K_(D) of less        than 10 pM as measured by surface plasmon resonance, and (ii) to        human IL-1beta with a K_(D) of less than 30 pM as measured by        surface plasmon resonance.    -   35. The antibody of any one of the preceding embodiments,        wherein an antibody Fab fragment of the antibody exhibits an        aggregation onset temperature of more than 70° C.    -   36. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein an antibody Fab fragment of the antibody        exhibits an aggregation onset temperature of more than 70° C.    -   37. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8, wherein an        antibody Fab fragment of the antibody exhibits an aggregation        onset temperature of more than 70° C.    -   38. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, (c) CDR-H3 comprising the amino acid sequence of SEQ        ID NO:15, (d) a human heavy chain framework with (i) FR1        comprising amino acid residues E2, G26, V28, and K30, (ii) FR3        comprising amino acid residues R66, R83, and K94, and a VL        domain comprising (e) CDR-L1 comprising the amino acid sequence        of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequence        of SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence        of SEQ ID NO: 8, and (h) a human light chain framework with (i)        FR1 comprising amino acid residue I2, (ii) FR2 comprising amino        acid residue Y49, (iii) FR3 comprising amino acid residues G57,        E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system, wherein an        antibody Fab fragment of the antibody exhibits an aggregation        onset temperature of more than 70° C.    -   39. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising within one pair of a VH and VL        domain: (i) a VH domain comprising amino acid residues E2, G26,        V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63,        I64, R66, R83, K94, D95, V96, F98, and D101, and (ii) a VL        domain comprising amino acid residues I2, Y27, W27a, S27c, S27d,        L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93,        H94, and Y96, wherein the numbering of the VH and VL domains is        according to the Kabat numbering system, wherein an antibody Fab        fragment of the antibody exhibits an aggregation onset        temperature of more than 70° C.    -   40. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11; and (b) a VL domain comprising an amino acid        sequence having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:12; wherein an antibody Fab fragment of        the antibody exhibits an aggregation onset temperature of more        than 70° C.    -   41. The antibody of any one of the preceding embodiments,        wherein an antibody Fab fragment of the antibody exhibits a        melting temperature of more than 80° C. as measured by dynamic        light scattering.    -   42. An antibody that specifically binds to human VEGF and to        human IL-1 beta, wherein an antibody Fab fragment of the        antibody exhibits a melting temperature of more than 80° C. as        measured by dynamic light scattering.    -   43. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8, wherein an        antibody Fab fragment of the antibody exhibits a melting        temperature of more than 80° C. as measured by dynamic light        scattering.    -   44. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, (c) CDR-H3 comprising the amino acid sequence of SEQ        ID NO:15, (d) a human heavy chain framework with (i) FR1        comprising amino acid residues E2, G26, V28, and K30, (ii) FR3        comprising amino acid residues R66, R83, and K94, and a VL        domain comprising (e) CDR-L1 comprising the amino acid sequence        of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequence        of SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence        of SEQ ID NO: 8, and (h) a human light chain framework with (i)        FR1 comprising amino acid residue I2, (ii) FR2 comprising amino        acid residue Y49, (iii) FR3 comprising amino acid residues G57,        E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system, wherein an        antibody Fab fragment of the antibody exhibits a melting        temperature of more than 80° C. as measured by dynamic light        scattering.    -   45. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising within one pair of a v VH and VL        domain: (i) a VH domain comprising amino acid residues E2, G26,        V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63,        I64, R66, R83, K94, D95, V96, F98, and D101, and (ii) a VL        domain comprising amino acid residues I2, Y27, W27a, S27c, S27d,        L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93,        H94, and Y96, wherein the numbering of the VH and VL domains is        according to the Kabat numbering system, wherein an antibody Fab        fragment of the antibody exhibits a melting temperature of more        than 80° C. as measured by dynamic light scattering.    -   46. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a (VL domain) comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11; and (b) a VL domain comprising an amino acid        sequence having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:12; wherein an antibody Fab fragment of        the antibody exhibits a melting temperature of more than 80° C.        as measured by dynamic light scattering.    -   47. The antibody of any one of the preceding embodiments,        wherein binding of an antibody Fab fragment of the antibody to        human VEGF inhibits binding of VEGF to VEGFR2 with an IC50 of        less than 50 nM as measured by surface plasmon resonance; and        wherein binding of an antibody Fab fragment of the antibody to        human IL-1beta inhibits binding of IL-1beta to IL-1betaR1 with        an IC50 of less than 30 nM as measured by surface plasmon        resonance.    -   48. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein binding of an antibody Fab fragment of        the antibody to human VEGF inhibits binding of VEGF to VEGFR2        with an IC50 of less than 50 nM as measured by surface plasmon        resonance; and wherein binding of an antibody Fab fragment of        the antibody to human IL-1beta inhibits binding of IL-1beta to        IL-1betaR1 with an IC50 of less than 30 nM as measured by        surface plasmon resonance.    -   49. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8, and wherein        binding of an antibody Fab fragment of the antibody to human        IL-1beta inhibits binding of IL-1beta to IL-1betaR1 with an IC50        of less than 30 nM as measured by surface plasmon resonance.    -   50. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, (c) CDR-H3 comprising the amino acid sequence of SEQ        ID NO:15, (d) a human heavy chain framework with (i) FR1        comprising amino acid residues E2, G26, V28, and K30, (ii) FR3        comprising amino acid residues R66, R83, and K94, and a VL        domain comprising (e) CDR-L1 comprising the amino acid sequence        of SEQ ID NO:16, (f) CDR-L2 comprising the amino acid sequence        of SEQ ID NO:17, (g) CDR-L3 comprising the amino acid sequence        of SEQ ID NO: 8, and (h) a human light chain framework with (i)        FR1 comprising amino acid residue I2, (ii) FR2 comprising amino        acid residue Y49, (iii) FR3 comprising amino acid residues G57,        E67, D68, and Q69, wherein the numbering of the VH and VL        domains is according to the Kabat numbering system, and wherein        binding of an antibody Fab fragment of the antibody to human        IL-1beta inhibits binding of IL-1beta to IL-1 betaR1 with an        IC50 of less than 30 nM as measured by surface plasmon        resonance.    -   51. An antibody that specifically binds to human VEGF and to        human IL-1beta, comprising within one pair of a VH and VL        domain: (i) a VH domain comprising amino acid residues E2, G26,        V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63,        I64, R66, R83, K94, D95, V96, F98, and D101, and (ii) a VL        domain comprising amino acid residues I2, Y27, W27a, S27c, S27d,        L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93,        H94, and Y96, wherein the numbering of the VH and VL domains is        according to the Kabat numbering system; and wherein binding of        an antibody Fab fragment of the antibody to human IL-1beta        inhibits binding of IL-1beta to IL-1betaR1 with an IC50 of less        than 30 nM as measured by surface plasmon resonance.    -   52. An antibody that specifically binds to human VEGF and to        human IL-1beta, wherein the antibody comprises a VH domain        comprising (a) CDR-H1 comprising the amino acid sequence of SEQ        ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ        ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of        SEQ ID NO:15, and a VL domain comprising (d) CDR-L1 comprising        the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising        the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3        comprising the amino acid sequence of SEQ ID NO:8,        comprising (a) a VH domain comprising an amino acid sequence        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:11; and (b) a VL domain comprising an amino acid        sequence having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:12; and wherein binding of an antibody Fab        fragment of the antibody to human IL-1beta inhibits binding of        IL-1 beta to IL-1betaR1 with an IC50 of less than 30 nM as        measured by surface plasmon resonance.    -   53. The antibody of any one of the preceding embodiments, which        is a monoclonal antibody.    -   54. The antibody of any one of the preceding embodiments, which        is an antibody fragment that binds to human VEGF and to human        IL-1beta.    -   55. The antibody of any one of the preceding embodiments,        wherein the antibody is bispecific.    -   56. The antibody of any one of the preceding embodiments,        wherein the antibody is a Fab fragment.    -   57. The antibody of any one of the preceding embodiments,        wherein the antibody is a bispecific antibody fragment.    -   58. The antibody of any one of the preceding embodiments,        wherein the antibody is a multispecific antibody.    -   59. An isolated nucleic acid encoding the antibody of any of        embodiments 1 to 58.    -   60. A host cell comprising the nucleic acid of embodiment 59.    -   61. An expression vector comprising the nucleic acid of        embodiment 61.    -   62. A method of producing an antibody that binds to human VEGF        and to human IL-1beta comprising culturing the host cell of        embodiment 60 so that the antibody is produced.    -   63. The method of embodiment 62, further comprising recovering        the antibody from the host cell.    -   64. An antibody produced by the method of embodiment 62 or 63.    -   65. A pharmaceutical formulation comprising the antibody of any        one of embodiments 1 to 58 and a pharmaceutically acceptable        carrier.    -   66. The antibody of any one of embodiments 1 to 58 for use as a        medicament.    -   67. The antibody of any one of embodiments 1 to 58 for use in        the treatment of a vascular disease.    -   68. The antibody of any one of embodiments 1 to 58 for use in        the treatment of an ocular vascular disease.    -   69. Use of the antibody of any one of embodiments 1 to 58 or the        pharmaceutical composition of embodiment 65 in the manufacture        of a medicament.    -   70. Use of the antibody of any one of embodiments 1 to 58 or the        pharmaceutical composition of embodiment 65 in the manufacture        of a medicament for inhibiting angiogenesis.    -   71. A method of treating an individual having a vascular disease        comprising administering to the individual an effective amount        of the antibody of one of embodiments 1 to 58 or the        pharmaceutical composition of embodiment 65.    -   72. A method of treating an individual having an ocular vascular        disease comprising administering to the individual an effective        amount of the antibody of one of embodiments 1 to 58 or        pharmaceutical composition of embodiment 65.    -   73. A method of inhibiting angiogenesis in an individual        comprising administering to the individual an effective amount        of the antibody of any of embodiments 1 to 58 or the        pharmaceutical composition of any of embodiments 65 to inhibit        angiogenesis.

Description of the Amino Acid Sequences

SEQ ID NO: 1 VH domain of 1HVL2.3EQLVESGGGLVKPGGSLRLSCAASGMVFSWNAMSWVRQAPGKGLEWVGSISPKGDHKYLNTKFIGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGFFDVWGQGTLVTVSS SEQ ID NO: 2 VL domain of 1HVL2.3AIYMHQEPSSLSASVGDRVTITCHGSYWLSNYLAWYQQKPGKAPKLLIYDASYRIIGVPSRFSGSGSHEDYTLTISSLQPEDFA TYYCQQYRYHPYTFGHGTKVEIKRSEQ ID NO: 3 H-CDR1 of 1HVL2.3 WNAMS SEQ ID NO: 4 H-CDR2 of 1HVL2.3SISPKGDHKYLNTKFIG SEQ ID NO: 5 H-CDR3 of 1HVL2.3 DIGFFDV SEQ ID NO: 6L-CDR1 of 1HVL2.3 HGSYWLSNYLA SEQ ID NO: 7 L-CDR2 of 1HVL2.3 DASYRIISEQ ID NO: 8 L-CDR3 of 1HVL2.3, 1HVL12.85, 1HVL5.15 and RO7200394QQYRYHPYT SEQ ID NO: 9 heavy chain of 1HVL2.3 Fab fragmentEQLVESGGGLVKPGGSLRLSCAASGMVFSWNAMSWVRQAPGKGLEWVGSISPKGDHKYLNTKFIGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYICNVNHKPSNTKVD KKVEPKSCDKTHT SEQ ID NO: 10light chain chain of 1HVL2.3 Fab fragmentAIYMHQEPSSLSASVGDRVTITCHGSYWLSNYLAWYQQKPGKAPKLLIYDASYRIIGVPSRFSGSGSHEDYTLTISSLQPEDFATYYCQQYRYHPYTFGHGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 11VH domain of 1HVL12.85 and RO7200394DEQLVESGGGLVKPGGSLRLSCAAEGMVFKWNDMSWVRQAPGKGLEWVGSISKKGDHKYLNTKFIGRFTISRDNEKDTLYLQMNSLRAEDTAVYYCAKDVGFFDIWGQGTLVTVSS SEQ ID NO: 12VL domain of 1HVL12.85 and RO7200394AIYMHQEPSSLSASVGDRVTITCHGSYWLSSLVAWYQQKPGKAPKLLIYDAKYKHLGVPSRFSGSKEDQEFTLTISSLQPEDFA TYYCQQYRYHPYTFGHGTKVEIKSEQ ID NO: 13 H-CDR1 of 1HVL12.85, 1HVL5.15 and RO7200394 WNDMSSEQ ID NO: 14 H-CDR2 of 1HVL12.85, 1HVL5.15 and RO7200394SISKKGDHKYLNTKFIG SEQ ID NO: 15H-CDR3 of 1HVL12.85, 1HVL5.15 and RO7200394 DVGFFDI SEQ ID NO: 16L-CDR1 of 1HVL12.85 and R07200394 HGSYWLSSLVA SEQ ID NO: 17L-CDR2 of 1HVL12.85, 1HVL5.15 and RO7200394 DAKYKHL SEQ ID NO: 18heavy chain of 1HVL12.85 Fab fragmentDEQLVESGGGLVKPGGSLRLSCAAEGMVFKWNDMSWVRQAPGKGLEWVGSISKKGDHKYLNTKFIGRFTISRDNEKDTLYLQMNSLRAEDTAVYYCAKDVGFFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYICNVNHKPSNTKV DKKVEPKSCDKTHT SEQ ID NO: 19light chain chain of 1HVL12.85 and RO7200394 Fab fragmentAIYMHQEPSSLSASVGDRVTITCHGSYWLSSLVAWYQQKPGKAPKLLIYDAKYKHLGVPSRFSGSKEDQEFTLTISSLQPEDFATYYCQQYRYHPYTFGHGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 20heavy chain of RO7200394 Fab fragment (SEQ ID NO:  19 withK196Q mutation) DEQLVESGGGLVKPGGSLRLSCAAEGMVFKWNDMSWVRQAPGKGLEWVGSISKKGDHKYLNTKFIGRFTISRDNEKDTLYLQMNSLRAEDTAVYYCAKDVGFFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHT SEQ ID NO: 21VH domain of 1HVL5.15 DETLVESGGGLVKPGGSLRLSCAAEGMVFKWNDMSWVRQAPGKGLEWVGSISKKGDHKYLNTKFIGRFTISRDNEKDTLYLQMNSLRAEDTAVYYCAKDVGFFDIWGQGTLVTVSS SEQ ID NO: 22 VL domain of 1HVL5.15AIYMHQEPSSLSASVGDRVTITCHGSYWLSSLMAWYQQKPGKAPKLLIYDAKYKHLGVPSRFSGSGSHEDYTLTISSLQPEDFA TYYCQQYRYHPYTFGHGTKVEIKSEQ ID NO: 23 L-CDR1 of 1HVL5.15 HGSYWLSSLMA SEQ ID NO: 24heavy chain of 1HVL5.15 Fab fragmentDETLVESGGGLVKPGGSLRLSCAAEGMVFKWNDMSWVRQAPGKGLEWVGSISKKGDHKYLNTKFIGRFTISRDNEKDTLYLQMNSLRAEDTAVYYCAKDVGFFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHT SEQ ID NO: 25light chain chain of 1HVL5.15 Fab fragmentAIYMHQEPSSLSASVGDRVTITCHGSYWLSSLMAWYQQKPGKAPKLLIYDAKYKHLGVPSRFSGSGSHEDYTLTISSLQPEDFATYYCQQYRYHPYTFGHGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 26 human VEGFMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTD SRCKARQLELNERTCRCDKPRRSEQ ID NO: 27 human IL1beta MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQT FQENDLSTFFPFIFEEEPIFFDTWDNE

EXAMPLES

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example 1

Generation of Bispecific Anti-VEGF/Anti-IL-1Beta Fab Fragment

A bispecific anti-VEGF/anti-IL-1beta Fab fragment was generated byindependent screening of monospecific antibodies that bind to VEGF andIL-1beta with non-overlapping paratopes and subsequent merging of theamino acid sequence into a biparatopic VH/VL pair that binds to VEGF andIL-1beta, by a method as described before, e.g. in WO2012/163520.

Two distinct phage display libraries of synthetic Fab fragments wereutilized, wherein in the first phage display library residues within theCDR-H1, CDR-H3 and CDR-L2 regions of the Fab fragments were diversified,and wherein in the second phage display library residues within theCDR-L1, CDR-L3 and CDR-H2 regions of the Fab fragments were diversified.In each library the other three CDR regions were kept non-diversified asinvariant dummy sequence. In both libraries the CH1 domain of the Fabfragments was fused via a linker to a truncated gene-III protein tofacilitate phage display.

The first library was enriched for binders against human IL-1beta, andthe second library was enriched for binders against human VEGF-A, byphage library panning. Following panning, plasmid minipreps weregenerated for both enriched pools of phagemid vectors. The miniprepswere digested with a restriction enzyme to excise the region encodingthe truncated gene-III protein and re-circularized by ligation to obtainpools of expression vectors encoding soluble Fab fragments that wereenriched for IL-1beta binders or for VEGF-A binders, respectively. Thesevector pools were transformed into TG1 E. coli cells and individualcolonies were picked and cultured for soluble expression of individualFab clones in microtiter plates. The supernatants comprising soluble Fabfragments were screened for binding to IL-1beta or VEGF-A using standardELISA methods, and TG1 clones producing specific binders were subjectedto DNA plasmid preparation and sequencing, to obtain pairs of VH and VLsequences specifically binding either to IL-1beta or to VEGF-A,respectively.

A pair of bispecific anti-VEGF/anti-IL-1beta VH and VL sequences wasdesigned in silico by (1) replacing the irrelevant VH residues 52b to 65in the VH sequence of an IL-1beta-specific Fab with selected VH residues52b to 65 of a VEGF-A-specific Fab, thus substituting CDR-H2 residuespotentially being part of the VEGF-A-specific paratope into the IL-1betabinder heavy chain, and (2) replacing the irrelevant VL residues 49 to57 in the VL sequence of a VEGF-A-specific Fab with selected VL residues49 to 57 of an IL-1beta-specific Fab, thus substituting CDR-L2 residuespotentially being part of the IL-1beta-specific paratope into the VEGF-Abinder light chain.

Example 2

Expression of Bispecific Anti-VEGF/Anti-IL-1Beta Fab Fragment 1HVL2.3

The resulting designed pair of bispecific anti-VEGF/anti-IL-1beta VH andVL sequences was synthesized and cloned into an E. coli expressionvector in frame with gene sequences encoding CH1 and Ckappa domains. Thevector was transformed into TG1 E. coli cells, and an individual colonywas cultured for soluble expression of the bispecific antibody Fabfragment. The bispecific antibody was purified from the TG1 culturesupernatant by affinity chromatography, and specific binding to bothIL-1beta and VEGF-A was verified.

Bispecific anti-VEGF/anti-IL-1beta antibody “1HVL2.3” was selected, andis characterized by a heavy chain of SEQ ID NO:9 and a light chain ofSEQ ID NO:10.

For further analyses anti-VEGF/anti-IL-1beta antibodies of the inventionwere transformed into and expressed from HEK293 cells by standardrecombinant methods.

Example 3

Characterization of Bispecific Anti-VEGF/Anti-IL-1Beta Fab Fragment1HVL2.3

Binding affinity, hydrophilcity and thermal stability of bispecificantibody 1HVL2.3 were assessed as follows:

VEGF Binding Kinetics as Assessed by Surface Plasmon Resonance (SPR):

An anti-His capturing antibody (GE Healthcare 28995056) was immobilizedto a Series S Sensor Chip C1 (GE Healthcare 29104990) using standardamine coupling chemistry resulting in a surface density of approximately500 resonance units (RU). As running and dilution buffer, HBS-P+ (10 mMHEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used. HumanVEGF121-His was captured to the surface with resulting ligand densitiesof approximately 10 and 20 RU, respectively. A dilution series of thebispecific anti-VEGF/anti-IL-1beta Fab fragment (1.2-100 nM, 1:3dilution) was successively injected for 90 s each, dissociation wasmonitored for 3600 s at a flow rate of 30 μl/min (single cyclekinetics). The surface was regenerated by injecting 10 mM Glycine pH 1.5for 60 s. Bulk refractive index differences were corrected bysubtracting blank injections and by subtracting the response obtainedfrom the control flow cell without captured human VEGF121. Curve fittingwas performed using the 1:1 Langmuir binding model within the Biacoreevaluation software. To provide more robust fitting, the Multiple Rmaxoption was chosen for global fitting using both ligand densities.

IL-1b Binding Kinetics as Assessed by Surface Plasmon Resonance (SPR):

An anti-Fab capturing antibody (GE Healthcare 28958325) was immobilizedto a Series S Sensor Chip C1 (GE Healthcare 29104990) using standardamine coupling chemistry resulting in a surface densitiy ofapproximately 500 resonance units (RU). The bispecificanti-VEGF/anti-IL-1beta Fab fragment was captured to the surface withresulting capture levels of approximately 20 RU. A dilution seriesranging from 0.74 to 60 nM (1:3 dilution) of either human IL-1 beta(PeproTech 200-01B) or cynomolgus IL-1 beta (Sino Biological 90010-CNAE)was injected for 90 s, dissociation was monitored for at least 600 s ata flow rate of 30 μl/min. The surface was regenerated by two consecutiveinjections of 10 mM Glycine pH 2.1 for 60 s each. Bulk refractive indexdifferences were corrected by subtracting blank injections and bysubtracting the response obtained from the control flow cell withoutcaptured bispecific anti-VEGF/anti-IL-1beta Fab fragment. Curve fittingwas performed using the 1:1 Langmuir binding model within the Biacoreevaluation software.

Hydrophobic Interaction Chromatography (HIC):

Apparent hydrophobicity was determined by injecting 20 μg of thebispecific anti-VEGF/anti-IL-1beta Fab fragment onto a HIC-Ether-5PW(Tosoh) column equilibrated with 25 mM Na-phosphate, 1.5 M ammoniumsulfate, pH 7.0. Elution was performed with a linear gradient from 0 to100% buffer B (25 mM Na-phosphate, pH 7.0) within 60 minutes. Retentiontimes were compared to protein standards with known hydrophobicity.

Thermal Stability:

Samples of the bispecific anti-VEGF/anti-IL-1 beta Fab fragment wereprepared at a concentration of 1 mg/mL in 20 mM Histidine/Histidinechloride, 140 mM NaCl, pH 6.0, transferred into an optical 384-wellplate by centrifugation through a 0.4 μm filter plate and covered withparaffine oil. The hydrodynamic radius is measured repeatedly by dynamiclight scattering on a DynaPro Plate Reader (Wyatt) while the samples areheated with a rate of 0.05° C./min from 25° C. to 80° C. Alternatively,samples were transferred into a 10 μL micro-cuvette array and staticlight scattering data as well as fluorescence data upon excitation witha 266 nm laser were recorded with an Optim1000 instrument (Avacta Inc.),while they were heated at a rate of 0.1° C./min from 25° C. to 90° C.

The aggregation onset temperature is defined as the temperature at whichthe hydrodynamic radius (DLS) or the scattered light intensity(Optim1000) starts to increase. The melting temperature is defined asthe inflection point in a fluorescence intensity vs. wavelength graph.

Results are shown in Tables 1 and 2.

TABLE 1 VEGF and IL-1beta binding kinetics of 1HVL2.3 as assessed by SPRhuman IL-1beta human VEGF 121 ka kd t1/2 K_(D) ka kd t1/2 K_(D) [1/Ms][1/s] [min] [pM] [1/Ms] [1/s] [min] [pM] 5.61E+06 2.56E−02 0.5 45651.75E+06 1.45E−05 796 8

TABLE 2 Thermal stability and hydrophobicity of 1HVL2.3 Tagg Tm (° C.)HIC (relative retention (° C.) DLS time) 75 (+/−1) 87 (+/−1) 0.58

Example 4

Improvement of Bispecific Anti-VEGF/Anti-IL-1Beta Fab Fragment 1HVL2.3

As illustrated above, the bispecific anti-VEGF/anti-IL-1beta Fabfragment 1HVL2.3, while being highly stable, exhibits an affinity toIL-1beta in the nanomolar range as well as significant hydrophobicity.For treatment of ocular vascular diseases, which requires injection ofthe therapeutic into the eye, it is desirable to provide the therapeuticwith a high affinity for the target antigen and in very highconcentrations to increase durability of the therapeutic effect and tominimize inconvienences for the patient. For the intended purpose it istherefore desired to increase affinity and to reduce hydrophobicity ofthe antibody to assure solubilty in isotonic buffer in highconcentrations.

Consequently, for clinical application the antibody required furtherimprovement, e.g. with respect to IL-1beta binding (particularly byimproving the off-rate) and reducing hydrophobicity. Several rounds ofmaturations were performed by introducing distinct amino acidsubstitutions in the VH and VL domain. During the maturations candidateantibodies derived from antibody 1HVL2.3 were screened and selectedbased on their desired properties with respect to yield, affinity,simultaneous antigen binding, hydrophilicity, stability, viscosity andother parameters.

Improved candidate antibodies 1HVL5.15, 1HVL12.85 and R07200394 wereselected from a plurality of tested candidate antibody molecules. Aminoacid sequences of those improved bispecific anti-VEGF/anti-IL-1beta Fabfragments are identified in Table 3.

TABLE 3 Amino acid sequences of bispecific anti-VEGF/anti-IL-1beta Fabfragments 1HVL2.3, 1HVL5.15, 1HVL12.85 and RO7200394 (the numbers referto the SEQ ID NOs as used herein) VH VL heavy chain light chain 1HVL2.31 2 9 10 1HVL5.15 21 22 24 25 1HVL12.85 11 12 18 19 RO7200394 11 12 2019FIGS. 2 and 3 illustrate an alignment of the variable heavy chaindomains and the variable light chain domains of the generated bispecificanti-VEGF/anti-IL-1beta Fab fragments. Numbering of the amino acidpositions within the VH and VL domains is according to the Kabatnumbering system. For simplicity, the numbering is included in theFigure, further illustrating framework and CDR amino acid positions.

Example 5

Antigen Binding Kinetics of Improved Bispecific Anti-VEGF/Anti-IL-1BetaFab Fragments

Binding kinetics to VEGF and IL-1beta for the candidate antibodies wereassessed as described in Example 3 using the indicated bispecificanti-VEGF/anti-IL-1beta Fab fragments (amino acid sequence asillustrated in Table 3). For comparison, antigen binding kinetics ofprior art anti-VEGF/anti-IL-1beta antibody 0032, a full length IgGantibody, as disclosed in WO2016/075034 are depicted.

Results of IL-1beta binding kinetics is shown in Table 4 and Table 5.

TABLE 4 Human IL-1beta binding kinetics of bispecific anti-VEGF/anti-IL-1beta antibodies as assessed by SPR (data for prior art antibody 0032from WO2016/075034) human IL-1beta antibody ka [1/Ms] kd [1/s] t1/2[min] K_(D) [pM] 0032 IgG 2.49E+06 3.05E−04 38 120 1HVL2.3 Fab 5.61E+062.56E−02 0.5 4565 1HVL5.15 Fab 1.66E+06 1.07E−04 109 ± 13* 59 ± 8*1HVL12.85 Fab 4.76E+06 1.35E−04 86 28 RO7200394 Fab 4.73E+06 1.02E−04114 21 *n = 4

TABLE 5 Cynomolgus IL-1beta binding kinetics of bispecific anti-VEGF/anti-IL-1beta antibodies as assessed by SPR cynomolgus IL-1beta antibodyka [1/Ms] kd [1/s] t1/2 [min] K_(D) [pM] 1HVL2.3 Fab 3.14E+06 1.82E−020.6 5794 1HVL5.15 Fab 2.60E+06 1.18E−04 98 45 1HVL12.85 Fab 3.34E+066.54E−05 177 20 RO7200394 Fab 2.97E+06 7.94E−05 146 27

Binding kinetics to IL-1beta of other species and related proteins wasassessed for antibodies 1HVL5.15 and 1HVL12.85 by SPR using the sameexperimental setup as described in Example 3. No binding was observedtowards rat IL-1beta, pig IL-1beta, human IL-1 alpha and human IL-1RA.Weak binding was observed towards murine and rabbit IL1-beta.

Results of VEGF binding kinetics is shown in Table 6.

TABLE 6 Human VEGF121 binding kinetics of bispecific anti-VEGF/anti-IL-1beta antibodies as assessed by SPR (data for prior art antibody 0032from WO2016/075034) human VEGF121 antibody ka [1/Ms] kd [1/s] t1/2 [min]K_(D) [pM] 0032 IgG 2.77E+04    <1E−06 <100 1HVL2.3 Fab 1.75E+061.45E−05 796 8 1HVL5.15 Fab 1.53E+06 1.47E−05 789 10 1HVL12.85 Fab2.50E+06 1.51E−05 764 6 RO7200394 Fab 2.85E+06 1.63E−05 707 6

Simultaneous antigen binding of the candidate antibodies to VEGF andIL-1beta was assessed as follows:

An anti-His capturing antibody (GE Healthcare 28995056) was immobilizedto a Series S Sensor Chip C1 (GE Healthcare 29104990) using standardamine coupling chemistry resulting in a surface densitiy ofapproximately 500 resonance units (RU). As running and dilution buffer,HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used.Human VEGF121-His was captured to the surface followed by consecutiveinjections of the candidate antibodies and IL-1beta. The surface wasregenerated by injecting 10 mM Glycine pH 1.5 for 60 s. Bulk refractiveindex differences were corrected by subtracting blank injections and bysubtracting the response obtained from the control flow cell withoutcaptured human VEGF121.

Simultaneous binding of the candidate antibodies to VEGF and IL-1betawas confirmed for all improved bispecific anti-VEGF/anti-IL-1beta Fabfragments, i.e. 1HVL5.15, 1HVL12.85 and R07200394. FIG. 4 illustratessimultaneous binding of anti-VEGF/anti-IL-1beta 1HVL12.85. FIG. 5illustrates simultaneous binding of anti-VEGF/anti-IL-1beta R07200394.

Simultaneous antigen binding of full length IgG prior art antibody 0032(WO2016/075034) was also assessed using the same experimental setup. Theresults are shown in FIG. 6 .

Example 6

Inhibition of Binding of VEGF and IL-1Beta to Respective Receptors

Receptor Inhibition Assay

Inhibition of binding of VEGF and IL-1beta to their respectivereceptors, hVEGFR2 and IL-1betaR1, in presence of candidate antibodyR07200394 (Fab fragment) was assessed as described below. Forcomparison, kinetics of prior art anti-VEGF/anti-IL-1beta antibody 0032(full length IgG), as disclosed in WO2016/075034, were assessed as well.

hVEGFR2 (R&D Systems 357-1(D) and IL-1bR1 (Sino Biological Inc.10126-H02H) were immobilized on different flow cells to a Series SSensor Chip CMS (GE Healthcare 29104988) using standard amine couplingchemistry resulting in surface densities of approximately 8000 and 20000resonance units (RU), respectively. As running and dilution buffer,HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used.For assessing VEGF-receptor binding inhibition, a final concentration of200 nM of RO7200394 or 400 nM antibody 0032 was preincubated with 50 nMVEGF121. For assessing IL-1beta-receptor binding inhibition, a finalconcentration of 200 nM of RO7200394 or 200 nM antibody 0032 was withpreincubated with 50 nM IL-1beta. Samples were diluted (1:2) in thecorresponding 50 nM VEGF121 or IL-1beta solution.The antibody/ligand mixtures were injected onto the VEGFR2 or IL-1R1surface for 60 s at a flow rate of 5 μl/min. After a dissociation phasefor 60 s, the VEGFR2 surface was regenerated by injecting 5 mM NaOH for60 s, whereas the IL-1R1 surface was regenerated by injecting 10 mMGlycine pH 3.0 followed by 5 mM NaOH for 60 s each. Bulk refractiveindex differences were corrected by subtracting blank injections and bysubtracting the response obtained from the blank control flow cell. Forevaluation, the binding response 5 seconds after inject end was taken.The derived response in RU was transformed to a binding responserelative to the initial signal corresponding to the ligand(s) withoutantibody. IC50 values were calculated using a 4 parameter logistic model(XLfit, ID Business Solutions Ltd.)Results are shown in Table 7 and Table 8; and FIG. 7A (VEGFR2 inhibitionin presence of antibody RO7200394), FIG. 7B (VEGFR2 inhibition inpresence of antibody 0032), FIG. 8A (IL-1R1 inhibition inhibition inpresence of antibody RO7200394) and FIG. 8B (IL-1R1 inhibitioninhibition in presence of antibody 0032).

TABLE 7 VEGR2 binding inhibition of bispecific anti-VEGF/anti-IL-1betaantibodies without (− IL-1beta) or in presence of IL-1beta (+ IL-1beta)IC50 IC50 antibody (−IL-1beta) (+IL-1beta) 0032 IgG 67 nM 62 nMRO7200394 Fab 44 nM 39 nM

TABLE 8 IL-1betaR1 binding inhibition of bispecificanti-VEGF/anti-IL-1beta antibodies without (− VEGF) or in presence ofVEGF (+ VEGF) IC50 IC50 antibody (−VEGF) (+VEGF) 0032 IgG 34 nM 34 nMRO7200394 Fab 22 nM 24 nMIt is demonstrated that binding of IL-1beta to the bispecificanti-VEGF/anti-IL-1beta Fab fragment of the invention does not interferewith inhibition of the VEGF/VEGFR2 interaction. Also, binding of VEGF tothe to the bispecific anti-VEGF/anti-IL-1beta Fab fragment of theinvention does not interfere with inhibition of the IL-1beta/IL-1betaR1interaction.VEGF Competition ELISAThe following buffers were used: PBS (1×PBS pH7.4); PBST (1×PBSsupplemented with 0.1% v/v Tween-20); PBST 1% BSA (PBST supplementedwith 1% BSA (Bovine Serum Albumin solution 30% from Sigma-Aldrich,A0336)); NaHCO₃(NaHCO₃ solution, pH 9.4, made from BupH Buffer Packs(ThermoScientific, 28382)); 2% MPBST (PBST supplemented with 2% (w/v)skimmed milk powder (Carl Roth, T145.3)).96-well-Plates (Maxisorp Nunc-Immoplates) were coated with 504/wellrhVEGFR-1-Fc (R&D #321-FL-050) at a final concentration of 1 μg/ml in200 mM NaHCO₃, pH 9.4 for 1 hour at room temperature.Meanwhile, antibody Fab fragments of different concentrations werepreincubated with VEGF for form an antibody Fab-VEGF premix as follows:A dilution series of antibody Fab fragment was prepared by adding 280 μlof a solution of antibody Fab fragments (409.6 nM antibody in PBST-1%BSA) into the wells of the first column of a round bottom 96-well PSplate. The individual wells of columns 2-12 of the same plate werefilled with 140 μl PBST-1% BSA. Subsequently, 1:2 dilutions wereperformed by transferring 140 μl of the antibody solution into the nextcolumn, thorough mixing and proceed with transferring 140 μl of thisdiluted antibody solution to the next colum. This is repeated untilcolumn 11. Excess of 140 μl were discarded so that all wells comprise140 μl. Colum 12 served as the control (blank). For preincubation withVEGF, round bottom 96-well PS plates were prefilled with 50 μl/well of a2 nM VEGF121 (Humanzyme, HZ-1206, Lot 0614-01) or a 2 nM VEGF165(Humanzyme, HZ-1153, Lot 0716-01) solution in PBST-1% BSA. 50 μl of thedilution series of the respective antibody Fab fragment were added tothe VEGF121, or VEGF165, respectively; mixed thoroughly and incubatedfor 1.5 hours at room temperature.

The rhVEGFR-1-Fc coated plates were washed two times with PBST andblocked for 45 min with 200 μl of 2% MPBST. After washing off the MPBSTsolution twice with PBST, 50 μl of the antibody Fab-VEGF premix wereadded to the plate and incubated for 1.5 hours at room temperature.Subsequently, the plates were washed twice with PBST. Then, 50 μl of asolution comprising biotinylated anti-VEGF antibody (R&D, BAF293; 1:2000dilution in PBST) and SA-HRP (KPL, 14-30-00; 1:2000 dilution in PBST)was added and incubated for 30 min at room temperature. After 6×washingwith PBST, 50 μl of TMB substrate solution (two-component HRP substrate(KPL, 34021); used at room temperature) was added and incubated for 30min at room temperature. 50 μl of 1N H₂SO₄ was added and absorbance wasread at 450 nm.

Results are shown in FIG. 9 and FIG. 10 and illustrate improvement ofVEGF-R1 binding inhibition of 1HVL12.85 and RO7200394 over 1HVL2.3.

Example 7

Biophysical Properties of Improved Bispecific Anti-VEGF/Anti-IL-1BetaFab Fragments (Stability and Hydrophobicity)

Indicated biophysical properties of the candidate antibodies wereassessed as described in Example 3 using the indicated bispecificanti-VEGF/anti-IL-1beta Fab fragments (amino acid sequence asillustrated in Table 3).

Table 9 illustrates the thermal stability and hydrophobicity of theanalysed antibodies. For comparison, the thermal stability of prior artanti-VEGF/anti-IL-1beta antibody 0032, a full length IgG antibody, asdisclosed in WO2016/075034 is included. Chromatograms from HIC are shownin FIG. 11 for the bispecific anti-VEGF/anti-IL-1beta Fab fragments andin FIG. 12 for prior art anti-VEGF/anti-IL-1beta IgG antibody 0032.

TABLE 9 Thermal stability and hydrophobicity of bispecificanti-VEGF/anti-IL- 1beta antibodies (data for prior art antibody 0032from WO2016/075034) HIC (relative Tagg Tm (° C.) retention antibody (°C.) DLS time) 0032 IgG 55 62.5 0.81 1HVL2.3 Fab 75 (+/−1) 87 (+/−1) 0.581HVL12.85 Fab 73 (+/−1) 85 (+/−1) 0.03 RO7200394 Fab 73 (+/−1) 85 (+/−1)0.04

Example 8 Biophysical Properties of Improved BispecificAnti-VEGF/Anti-IL-1Beta Fab Fragments (Dynamic Viscosity)

The viscosity of the candidate antibodies was assessed as follows usingthe indicated bispecific anti-VEGF/anti-IL-1beta Fab fragments (aminoacid sequence as illustrated in Table 3):

Viscosity was measured with the latex-bead DLS method as describedbefore (He F et al.; Anal Biochem. 2010 Apr. 1; 399(1):141-3). In brief,samples were concentrated to >200 mg/mL (based on material availability)with centrifugal concentration devices, e.g. Amicon Ultra—0.5 mLCentrifugal Filters, Ultracel—10K, Cat.-No. UFC501096.

A dilution series from approximately 10 mg/mL to the maximal feasibleconcentration was prepared and Polysorbate 20 and beads (Nanosphere SizeStandards, Nom Diam: 300 nm, 1% solids, ThermoFisher Cat. No. 3300A)were added to a final concentration of 0.02% (PS20) and 0.03% (w/w,beads), respectively.

A small aliquot of these samples was centrifuged for 1 minute at maximumspeed before the protein concentration was determined by UV280absorption.

The remaining sample was transferred to an optical 384-well plate,covered with a layer of paraffin oil to prevent evaporation, and DLSdata were recorded at the indicated temperature. From the DLS data forapparent hydrodynamic radius of the latex beads, the viscosity of thesolution was calculated as described in He F et al.; supra. Theviscosity at the highest concentration measured is reported in Table 10.Results are also shown in FIG. 13 (1HVL12.85) and FIG. 14 (RO7200394).

TABLE 10 Viscosity of bispecific anti-VEGF/anti-IL-1beta antibodies at15° C. antibody Viscosity 1HVL12.85 Fab 18.4 cP @ 195 mg/ml RO7200394Fab 14.6 cP @ 210 mg/ml

Results indicate that antibodies of the invention may be formulated inhigh concentrations comprising a viscosity below the acceptableviscosity limit for syringeabilty, which is up to 30 cP. While bothtestes antibodies are shown to be be highly concentratable, the effectis more prominent in the R07200394 antibody.

In consequence, the antibodies of the invention are highly suitable forocular application as they allow for provision of a high molar dose in alimited injection volume, which when combined with high potency resultsin a high durability and consequently, a reduced dosing frequency, whichis desirable to reduce difficulties on the patient's side.

Example 9 Chemical Stability of Improved BispecificAnti-VEGF/Anti-IL-1Beta Fab Fragment

Chemical Degradation Test:

Antibody samples were formulated in 20 mM His/HisCl, 140 mM NaCl, pH6.0, and were split into three aliquots: one aliquot was re-bufferedinto PBS, respectively, and two aliquots were kept in the originalformulation. The PBS aliquot and one His/HisCl aliquot were incubatedfor 2 weeks (2 w) at 40° C. (His/NaCl) or 37° C. (PBS) in 1 mg/ml, thePBS sample was incubated further for total 4 weeks (4w). The thirdcontrol aliquot sample was stored at −80° C. After incubation ended,samples were analyzed for relative active concentration (Biacore; activeconcentration of both stressed aliquots of each binder is normalized tounstressed 4° C. aliquot), aggregation (SEC) and fragmentation(capillary electrophoresis or SDS-PAGE) and compared with the untreatedcontrol.

Binding Activity after Stress was Assessed as Follows:

Anti-Fab capturing antibody (GE Healthcare 28958325) was immobilized ona Series S Sensor Chip CMS (GE Healthcare 29104988) using standard aminecoupling chemistry resulting in a surface density of 4000-6000 resonanceunits (RU). As running and dilution buffer, HBS-P+ (10 mM HEPES, 150 mMNaCl pH 7.4, 0.05% Surfactant P20) was used. Antibodyanti-VEGF/anti-IL-1beta antibody 1HVL12.85 having a concentration of 2μg/ml was injected for 60 s at a flow rate of 5 μl/min. HuVEGF121 (inhouse preparation) or huIL-1 beta (Peprotech 200-01B) at a concentrationof 2 μg/ml each was injected for 60 s, dissociation was monitored for 60s at a flow rate of 5 μl/min. The surface was regenerated by twoconsecutive injections of 10 mM Glycine pH 2.1 for 60 s each. Bulkrefractive index differences were corrected by subtracting blankinjections and by subtracting the response obtained from the blankcontrol flow cell. For evaluation, the binding response 5 seconds afterinject end was taken. To normalize the binding signal, the VEGF or IL-1beta binding response was divided by the anti-Fab response. The relativeactive concentration was calculated by referencing each temperaturestressed sample to the corresponding, non-stressed sample.Results are shown in Tables 11 and 12.

TABLE 11 Binding activity after stress for anti-VEGF/anti-IL-1betaantibody 1HVL12.85 (amino acid sequence see Table 3) Binding activityafter Binding activity after stress IL-1beta stress VEGF stressconditions [% binding] [% binding] 2 w/40° C./pH 6.0 98 98 2 w/37° C./pH7.4 98 96 4 w/40° C./pH 6.0 97 95 4 w/37° C./pH 7.4 97 94

TABLE 12 Molecular integrity after stress for anti-VEGF/anti-IL-1betaantibody 1HVL12.85 (amino acid sequence see Table 3) AggregationFragmentation stress conditions [% aggregates SEC] [% fragments CE-SDS]2 w/40° C./pH 6.0 0.98 1.68 2 w/37° C./pH 7.4 1.94 3.39

Example 10 Structural Analysis of Improved BispecificAnti-VEGF/Anti-IL-1Beta Fab Fragment 1HVL5.15

Structural analysis of anti-VEGF/anti-IL-1beta Fab fragment 1HVL5.15 wasperformed by x-ray crystallography as follows:

Complex Formation and Crystallization of the Ternary ComplexIL1β-VEGF121—Fab 1HVL5.15

For complex formation, antibody 1HVL5.15 Fab fragment and human IUD(Peprotech) were mixed in a 1:1.1 molar ratio. After incubation for 16hours overnight at 4° C., human VEGF121 (in house preparation) was addedto obtain a ternary complex which was concentrated to 10 mg/ml. Initialcrystallization trials were performed in sitting drop vapor diffusionsetups at 21° C. First micro-crystals appeared within 4 days out of 1.4Msodium malonate. Subsequent seeding experiments yielded crystals out of0.1 M sodium cacodylate pH 5.5, 0.1 M calcium acetate, 12% PEG8000. Thecrystals were directly harvested from the screening plate without anyfurther optimization steps.Data Collection and Structure DeterminationFor data collection crystals were flash cooled at 100K in precipitantsolution with addition of 15% ethylene glycol as cryoprotectant.Diffraction data were collected at a wavelength of 1.0000 Å using aPILATUS 6M detector at the beamline X10SA of the Swiss Light Source(Villigen, Switzerland). Data have been processed with XDS (Kabsch, W.Acta Cryst. D66, 133-144 (2010)) and scaled with SADABS (BRUKER). Thecrystals belong to the space group C222₁ with cell axes of a=177.97 Å,b=286.70 Å, c=105.39 Å, α=β=γ=90° and diffract to a resolution of 2.97Å. The structure was determined by molecular replacement with PHASER(McCoy, A. J. et al. J. Appl. Cryst. 40, 658-674 (2007)) using thecoordinates of a related in house structures of a Fab fragment, IL1β andpdb entry 1MKK for VEGF as search models. Programs from the CCP4 suite(Collaborative Computational Project, Number 4 Acta Cryst. D50, 760-763(1994)) and Buster (Bricogne, G., et al. (2011). Buster version 2.9.5Cambridge, United Kingdom: Global Phasing Ltd) have been used forrefinement of the structure. Manual rebuilding of protein usingdifference electron density was done with COOT (Emsley, P., et al. ActaCryst D66, 486-501 (2010)). Data collection and refinement statisticsare summarized in Table 13. All graphical presentations were preparedwith PYMOL (DeLano Scientific, Palo Alto, C A, 2002). The structure wasanalyzed with the program CONTACT from the CCP4 suite (CollaborativeComputational Project, Number 4 Acta Cryst. D50, 760-763 (1994)) toidentify paratope and epitope residues using a contact distance maximumof 4 Å.

TABLE 13 Data collection and structure refinement statistics (x-raycrystallography) Data Collection Wavelength (Å) 1.0 Resolution¹ (Å)49.76-2.97 (3.07-2.97)  Space group C222₁ Unit cell (Å, °) 177.97,286.70,105.39, 90° Unique reflections 55924 (5206)  Multiplicity 7.60(7.76) Completeness (%) 99.9 (99.9) Mean I/σ (I) 6.53 (0.84) R-meas 0.25(0.93) CC1/2 0.999 (0.297) Refinement Resolution¹ (Å) 49.76-2.97(3.02-2.97)  Reflections used in refinement 55851 (2753)  Reflectionsused for R-free 2595 (109)  R-work ³ 0.204 (0.262) R-free ⁴ 0.252(0.287) Number of atoms 10540 Protein residues 577 RMS bonds (Å) 0.010RMS angles (°) 1.40 Ramachandran favored (%) 95.32 Ramachandran outliers(%) 0.15 Rotamer outliers (%) 0.84 Clashscore 9.03 Average B-factor (Å²)72.98 protein 72.98 ¹Values in parentheses refer to the highestresolution bins. ²R_(merge) = Σ|I − <I>|/ΣI where I is intensity. ³R_(work) = Σ|F_(o) − <F_(c)>|/ΣF_(o) where F_(o) is the observed andF_(c) is the calculated structure factor amplitude. ⁴ R_(free) wascalculated based on 5% of the total data omitted during refinement.

Amino acid residues in contact with the respective antigens, VEGF andIL-1beta, were identified from the crystal structure of the bispecificanti-VEGF/anti-IL-1beta Fab fragment 1HVL5.15 in complex. Anillustration of the position of paratope amino acid residues within theVH and VL domains is depicted in FIG. 2 and FIG. 3 .

Amino acids from light chain CDR1 and CDR3 as well as heavy chain CDR2contribute to the VEGF paratope. The VEGF paratope does not compriseamino acids from light chain CDR2, heavy chain CDR1 and heavy chainCDR3. The IL-1beta paratope does not comprise amino acids from lightchain CDR2.

The amino acid residues identified to contribute to antigen binding areidentified in Table 14 (for the variable heavy chain domain amino acidresidues) and Table 15 (for the variable light chain domain amino acidresidues). Amino acid positions are numbered according to the Kabatnumbering system (the same numbering is used in FIGS. 2 and 3 ). Aminoacids positions involved in antigen binding are identified by theirKabat position in the VH or VL domain (see also the numbering in FIGS. 2and 3 ).

TABLE 14 Variable heavy chain amino acid residues involved in antigenbinding as identified by crystal structure analysis of bispecificanti-VEGF/anti-IL-1beta antibody 1HVL5.15 VH VEGF IL-1beta FR1 — 2, 26,28, 30 H-CDR1 — 31, 35b, 35c FR2 — — H-CDR2 55, 56, 58, 61, 62, 63, 6452a FR3 66, 83 94 H-CDR3 — 95, 96, 98, 101 FR4 — —

TABLE 15 Variable light chain amino acid residues involved in antigenbinding as identified by crystal structure analysis of bispecific anti-VEGF/anti-IL-1beta antibody 1HVL5.15 VL VEGF IL-1beta FR1 2 — L-CDR1 27,27a, 27c, 27d 32 FR2 — 49 L-CDR2 — 50, 53, 54, 56 FR3 67, 68, 69 57L-CDR3 92, 93, 94, 96 91 FR4 — —

What is claimed is:
 1. A method for decreasing the rate of oculardisease progression in an individual having the ocular disease,comprising administering to the individual a therapeutically effectiveamount of an antibody or antigen-binding fragment thereof that binds tohuman VEGF and to human IL-1beta, wherein the antibody comprises a VEGFparatope and an IL-1beta paratope within one cognate pair of a variablelight chain domain (VL domain) and a variable heavy chain domain (VHdomain), wherein the VEGF paratope comprises amino acid residues fromCDR-H2, CDR-L1 and CDR-L3 of the antibody that binds to human VEGF,wherein the IL-1beta paratope comprises amino acid residues from theCDR-H1, CDR-H3 and CDR-L2 of the antibody that binds to human IL-1beta,wherein the antibody or antigen-binding fragment thereof comprises a VHdomain comprising (a) CDR-H1 comprising the amino acid sequence of SEQID NO: 13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15,and a VL domain comprising (d) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 16, (e) CDR-L2 comprising the amino acid sequence of SEQID NO: 17, and (f) CDR-L3 comprising the amino acid sequence of SEQ IDNO: 8, and wherein the administering decreases the rate of oculardisease progression in the individual, wherein the ocular disease isselected from the group consisting of age-related macular degeneration(AMD), geographic atrophy (GA), macular degeneration, macular edema,diabetic macular edema (DME), retinopathy, and diabetic retinopathy(DR).
 2. The method of claim 1, wherein the AMD is wet AMD, dry AMD,intermediate AMD, or advanced AMD.
 3. The method of claim 1, furthercomprising administering to the individual at least one additionaltherapeutic agent.
 4. The method of claim 1, wherein the antibody orantigen-binding fragment thereof is a Fab fragment of the antibody whichbinds (i) to human VEGF121 with a K_(D) of less than 10 pM as measuredby surface plasmon resonance, and (ii) to human IL-1beta with a KD ofless than 30 pM as measured by surface plasmon resonance.
 5. The methodof claim 1, wherein the antibody or antigen-binding fragment thereof isa Fab fragment of the antibody wherein the Fab fragment exhibits amelting temperature of more than 80° C. as measured by dynamic lightscattering.
 6. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises (a) a VH domain comprising anamino acid sequence having at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:11; and (b) a VL domain comprising an aminoacid sequence having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:12.
 7. The method of claim 1, wherein the antibodyor antigen-binding fragment thereof comprises (a) a VH domain comprisingthe amino acid sequence of SEQ ID NO:11 with up to 15 amino acidsubstitutions; and (b) a variable light chain domain comprising theamino acid sequence of SEQ ID NO:12 with up to 15 amino acidsubstitutions.
 8. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises the heavy chain sequence ofSEQ ID NO:18 and the light chain sequence of SEQ ID NO:19.
 9. The methodof claim 1, wherein the antibody or antigen-binding fragment thereof isa Fab that comprises the heavy chain sequence of SEQ ID NO:18 and thelight chain sequence of SEQ ID NO:19.
 10. The method of claim 1, whereinthe antibody or antigen-binding fragment thereof comprises the VH domainsequence of SEQ ID NO:11 and the VL domain sequence of SEQ ID NO:12. 11.The method of claim 10, wherein the ocular disease is selected from thegroup consisting of age-related macular degeneration (AMD), geographicatrophy (GA), macular degeneration, macular edema, diabetic macularedema (DME), retinopathy, and diabetic retinopathy (DR).
 12. The methodof claim 11, wherein the AMD is wet AMD, dry AMD, intermediate AMD, oradvanced AMD.
 13. The method of claim 10, further comprisingadministering to the individual at least one additional therapeuticagent.
 14. The method of claim 1, wherein the antibody orantigen-binding fragment thereof is a Fab that comprises the VH domainsequence of SEQ ID NO:11 and the VL domain sequence of SEQ ID NO:12. 15.A method for decreasing the rate of ocular disease progression in anindividual having the ocular disease, comprising administering to theindividual a therapeutically effective amount of an antibody orantigen-binding fragment thereof that binds to human VEGF and to humanIL-1beta, wherein the antibody comprises a VEGF paratope and an IL-1betaparatope within one cognate pair of a variable light chain domain (VLdomain) and a variable heavy chain domain (VH domain), wherein the VEGFparatope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 ofthe antibody that binds to human VEGF, wherein the IL-1beta paratopecomprises amino acid residues from the CDR-H1, CDR-H3 and CDR-L2 of theantibody that binds to human IL-1beta, wherein the antibody orantigen-binding fragment thereof comprises a VH domain comprising (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 14, and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 15, and a VL domaincomprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and(f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, andwherein the administering decreases the rate of ocular diseaseprogression in the individual, wherein the ocular disease is selectedfrom the group consisting of age-related macular degeneration (AMD),geographic atrophy (GA), macular degeneration, macular edema, diabeticmacular edema (DME), retinopathy, and diabetic retinopathy (DR).
 16. Themethod of claim 15, wherein the antibody or antigen-binding fragmentthereof comprises the heavy chain sequence of SEQ ID NO:24 and the lightchain sequence of SEQ ID NO:25.
 17. The method of claim 15, wherein theantibody or antigen-binding fragment thereof is a Fab that comprises theheavy chain sequence of SEQ ID NO:24 and the light chain sequence of SEQID NO:25.
 18. A method for decreasing the rate of ocular diseaseprogression in an individual having the ocular disease, comprisingadministering to the individual a therapeutically effective amount of anantibody or antigen-binding fragment thereof that binds to human VEGFand to human IL-1beta, wherein the antibody comprises a VEGF paratopeand an IL-1beta paratope within one cognate pair of a variable lightchain domain (VL domain) and a variable heavy chain domain (VH domain),wherein the VEGF paratope comprises amino acid residues from CDR-H2,CDR-L1 and CDR-L3 of the antibody that binds to human VEGF, wherein theIL-1beta paratope comprises amino acid residues from the CDR-H1, CDR-H3and CDR-L2 of the antibody that binds to human IL-1beta, wherein theantibody or antigen-binding fragment thereof comprises a VH domaincomprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and a VLdomain comprising (d) CDR-L1 comprising the amino acid sequence of SEQID NO: 16, (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8,and wherein the administering decreases the rate of ocular diseaseprogression in the individual, wherein the ocular disease is selectedfrom the group consisting of age-related macular degeneration (AMD),geographic atrophy (GA), macular degeneration, macular edema, diabeticmacular edema (DME), retinopathy, and diabetic retinopathy (DR).
 19. Themethod of claim 18, wherein the antibody or antigen-binding fragmentthereof comprises the heavy chain sequence of SEQ ID NO:20 and the lightchain sequence of SEQ ID NO:19.
 20. The method of claim 18, wherein theantibody or antigen-binding fragment thereof is a Fab that comprises theheavy chain sequence of SEQ ID NO:20 and the light chain sequence of SEQID NO:19.
 21. A method for inhibiting angiogenesis in the eye of anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of an antibody or antigen-bindingfragment thereof that binds to human VEGF and to human IL-1beta, whereinthe antibody comprises a VEGF paratope and an IL-1beta paratope withinone cognate pair of a variable light chain domain (VL domain) and avariable heavy chain domain (VH domain), wherein the VEGF paratopecomprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of theantibody, wherein the IL-1beta paratope comprises amino acid residuesfrom the CDR-H1, CDR-H3 and CDR-L2 of the antibody, wherein the antibodyor antigen-binding fragment thereof comprises a VH domain comprising (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO:13, (b) CDR-H2comprising the amino acid sequence of SEQ ID NO:14, and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO:15, and a VL domaincomprising (d) CDR-L1 comprising the amino acid sequence of SEQ IDNO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:17,and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8. 22.The method of claim 21, further comprising administering to theindividual at least one additional therapeutic agent.
 23. The method ofclaim 21, wherein the antibody or antigen-binding fragment thereof is aFab fragment of the antibody which binds (i) to human VEGF121 with a KDof less than 10 pM as measured by surface plasmon resonance, and (ii) tohuman IL-1beta with a KD of less than 30 pM as measured by surfaceplasmon resonance.
 24. The method of claim 21, wherein the antibody orantigen-binding fragment thereof is a Fab fragment of the antibodywherein the Fab fragment exhibits a melting temperature of more than 80°C. as measured by dynamic light scattering.
 25. The method of claim 21,wherein the antibody or antigen-binding fragment thereof comprises (a) aVH domain comprising an amino acid sequence having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:11; and (b) a VL domaincomprising an amino acid sequence having at least 90% sequence identityto the amino acid sequence of SEQ ID NO:12.
 26. The method of claim 21,wherein the antibody or antigen-binding fragment thereof comprises (a) aVH domain comprising the amino acid sequence of SEQ ID NO:11 with up to15 amino acid substitutions; and (b) a variable light chain domaincomprising the amino acid sequence of SEQ ID NO:12 with up to 15 aminoacid substitutions.
 27. The method of claim 21, wherein the antibody orantigen-binding fragment thereof comprises the heavy chain sequence ofSEQ ID NO:18 and the light chain sequence of SEQ ID NO:19.
 28. Themethod of claim 21, wherein the antibody or antigen-binding fragmentthereof is a Fab that comprises the heavy chain sequence of SEQ ID NO:18and the light chain sequence of SEQ ID NO:19.
 29. The method of claim21, wherein the antibody or antigen-binding fragment thereof comprisesthe VH domain sequence of SEQ ID NO:11 and the VL domain sequence of SEQID NO:12.
 30. The method of claim 21, wherein the antibody orantigen-binding fragment thereof is a Fab that comprises the VH domainsequence of SEQ ID NO:11 and the VL domain sequence of SEQ ID NO:12. 31.A method for inhibiting angiogenesis in the eye of an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of an antibody or antigen-binding fragment thereof thatbinds to human VEGF and to human IL-1beta, wherein the antibodycomprises a VEGF paratope and an IL-1beta paratope within one cognatepair of a variable light chain domain (VL domain) and a variable heavychain domain (VH domain), wherein the VEGF paratope comprises amino acidresidues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, wherein theIL-1beta paratope comprises amino acid residues from the CDR-H1, CDR-H3and CDR-L2 of the antibody, wherein the antibody or antigen-bindingfragment thereof comprises a VH domain comprising (a) CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:13, (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO:14, and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO:15, and a VL domain comprising (d) CDR-L1comprising the amino acid sequence of SEQ ID NO:23, (e) CDR-L2comprising the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3comprising the amino acid sequence of SEQ ID NO:8.
 32. The method ofclaim 31, wherein the antibody or antigen-binding fragment thereofcomprises the heavy chain sequence of SEQ ID NO:24 and the light chainsequence of SEQ ID NO:25.
 33. The method of claim 31, wherein theantibody or antigen-binding fragment thereof is a Fab that comprises theheavy chain sequence of SEQ ID NO:24 and the light chain sequence of SEQID NO:25.
 34. A method for inhibiting angiogenesis in the eye of anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of an antibody or antigen-bindingfragment thereof that binds to human VEGF and to human IL-1beta, whereinthe antibody comprises a VEGF paratope and an IL-1beta paratope withinone cognate pair of a variable light chain domain (VL domain) and avariable heavy chain domain (VH domain), wherein the VEGF paratopecomprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of theantibody, wherein the IL-1beta paratope comprises amino acid residuesfrom the CDR-H1, CDR-H3 and CDR-L2 of the antibody, wherein the antibodyor antigen-binding fragment thereof comprises a VH domain comprising (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO:5, and a VL domaincomprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:6,(e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:7, and (f)CDR-L3 comprising the amino acid sequence of SEQ ID NO:8.
 35. The methodof claim 34, wherein the antibody or antigen-binding fragment thereofcomprises the heavy chain sequence of SEQ ID NO:20 and the light chainsequence of SEQ ID NO:19.
 36. The method of claim 34, wherein theantibody or antigen-binding fragment thereof is a Fab that comprises theheavy chain sequence of SEQ ID NO:20 and the light chain sequence of SEQID NO:19.